U.S. patent application number 15/744053 was filed with the patent office on 2018-09-27 for her2 binding proteins based on di-ubiquitin muteins.
This patent application is currently assigned to Navigo Proteins GmbH. The applicant listed for this patent is Navigo Proteins GmbH. Invention is credited to Eva Bosse-Doenecke, Erik Fiedler, Manja Gloser, Ulrich Haupts, Florian Settele, Madlen Zwarg.
Application Number | 20180273636 15/744053 |
Document ID | / |
Family ID | 53776345 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273636 |
Kind Code |
A1 |
Settele; Florian ; et
al. |
September 27, 2018 |
HER2 BINDING PROTEINS BASED ON DI-UBIQUITIN MUTEINS
Abstract
The present invention relates to new Her2 binding molecules
based on di-ubiquitin muteins. The invention further refers to Her2
binding proteins optionally fused or conjugated to a moiety
modulating pharmacokinetics or to a therapeutically or
diagnostically active component. The invention further relates to
the use of these Her2 binding proteins in medicine, preferably for
use in the diagnosis or treatment of cancer.
Inventors: |
Settele; Florian;
(Halle/Saale, DE) ; Zwarg; Madlen; (Halle/Saale,
DE) ; Gloser; Manja; (Teutschenthal, DE) ;
Bosse-Doenecke; Eva; (Halle/Saale, DE) ; Fiedler;
Erik; (Halle/Saale, DE) ; Haupts; Ulrich;
(Halle/Saale, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Navigo Proteins GmbH |
Halle/Saale |
|
DE |
|
|
Assignee: |
Navigo Proteins GmbH
Halle/Saale
DE
|
Family ID: |
53776345 |
Appl. No.: |
15/744053 |
Filed: |
July 19, 2016 |
PCT Filed: |
July 19, 2016 |
PCT NO: |
PCT/EP2016/067207 |
371 Date: |
January 11, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/94 20130101;
C07K 16/32 20130101; C07K 2318/20 20130101; C07K 16/2863 20130101;
A61P 35/00 20180101; C07K 14/47 20130101; C07K 2319/00 20130101;
C07K 2317/92 20130101; C07K 2317/31 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 20, 2015 |
EP |
EP15177548.3 |
Claims
1. A Her2 binding protein comprising an amino acid sequence that is
at least 85% identical to SEQ ID NO: 4, wherein the Her2 binding
protein comprises substitutions of 12-14 amino acids at a position
selected from the group consisting of R42, I44, H68, V70, R72, L73,
R74, K82, L84, Q138, K139, E140, S141, and T142 of SEQ ID NO: 4 and
has a binding affinity (K.sub.D) of less than 700 nM for Her2.
2. The Her2 binding protein of claim 1, wherein the amino acid
sequence further comprises 1-6 additional substitutions (as
compared to SEQ ID NO: 4.
3. The Her2 binding protein of claim 1, wherein as compared to SEQ
ID NO: 4: position R42 is substituted by a polar amino acid,
position I44 is substituted by a hydrophobic or polar amino acid,
position H68 is substituted by an aromatic amino acid, position V70
is substituted by an aromatic amino acid, position R72 is
substituted by a polar or aromatic amino acid, position L73 is
substituted by any amino acid but not basic or acidic amino acid,
position R74 is substituted by an aromatic, basic or polar amino
acid, position K82 is substituted by any amino acid but not basic
or acidic amino acid, position L84 is substituted by a basic or
acidic amino acid, position Q138 is substituted by a basic or
acidic or polar amino acid, position K139 is substituted by acidic
or hydrophobic amino acid or Glycine, position E140 is substituted
by an aromatic amino acid, position S141 is substituted by a
hydrophobic or polar or basic amino acid, and/or position T142 is
substituted by a hydrophobic or polar amino acid.
4. The Her2 binding protein of claim 3, wherein the substitutions
are selected from the group consisting of R42T, R42S, R42L, I44A,
I44V, I44S, I44T, H68W, H68Y, H68F, V70Y, V70W, R72T, R72F, R72G,
R72Y, L73W, L73S, L73V, L731, R74Y, R74S, R74N, R74K, K82T, K82L,
K82N, K821, K82Y, L84H, L84D, L84E, L84S, Q138S, Q138R, Q138E,
K139E, K139G, K139L, E140W, S141A, S141R, T142I, T142L, and/or and
T142N, and combinations thereof.
5. The Her2 binding protein of claim 4, wherein the substitutions
are selected from the group consisting of Q138S, K139E, E140W,
S141A, and T142I; orQ138R, K139G, E140W, and T142L; or Q138E,
K139L, E140W, S141R, and T142N.
6. The Her2 binding protein of claim 4, wherein the substitutions
are selected from the group consisting of R42T, I44A, H68W, V70Y,
R72T, L73W, R74Y, K82T, and L84H.
7. The Her2 protein of claim 4, wherein the amino acids
substitutions are selected from the group consisting of R42S, I44V,
H68Y, V70Y, R72F, L73S, K82L, and L84D.
8. The Her2 binding protein of claim 1, wherein the amino acid
sequence is selected from the group consisting of SEQ ID NOs:
5-38.
9. The Her2 binding protein of claim 1, wherein the Her2 binding
protein binds to a Her2 epitope that is different from or that does
not overlap with that to which monoclonal antibody Trastuzumab
binds.
10. The Her2 binding protein of claim 1, wherein the Her2 binding
protein is conjugated to or fused to at least one additional
molecule, and further wherein the at least one additional molecule
is selected from the group consisting of a pharmacokinetic
modulating moiety, a therapeutically active component, diagnostic
component.
11. The Her2 binding protein of claim 10, wherein the at least one
additional molecule comprises a monoclonal antibody with
specificity for EGFR.
12. A method for diagnosing or treating a disease or disorder
associated with Her2 expression, the method comprising
administering to a subject in need thereof a diagnostically or
therapeutically effective dose of the Her2 binding protein of claim
1.
13. A nucleic acid molecule encoding the Her2 binding protein-as of
claim 1.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A host cell or a non-human host comprising the Her2 binding
protein of claim 1 or a nucleic acid molecule encoding the Her2
binding protein of claim 1.
16. A composition comprising the Her2 binding protein of claim 1
and a pharmaceutically acceptable carrier.
17. A method for producing a Her2 binding protein of claim 1, the
method comprising culturing a host cell comprising a nucleic acid
molecule encoding the Her2 binding protein of claim 1 under
suitable conditions whereby the host cell expresses the Her2
binding protein of claim 1.
18. The Her2 binding protein of claim 10, wherein the
pharmacokinetic modulating moiety is selected from the group
consisting of a polyethylene glycol, a human serum albumin, an
albumin-binding peptide, an immunoglobulin molecule or a fragment
thereof, and a polysaccharide.
19. The Her2 binding protein of claim 10, wherein the
therapeutically active component is selected from the group
consisting of a monoclonal antibody or a fragment thereof, a
cytokine, a chemokine, a cytotoxic compound, an enzyme, and a
radionuclide.
20. The Her2 binding protein of claim 10, wherein the diagnostic
component is selected from the group consisting of a fluorescent
compound, a photosensitizer, a tag, an enzyme, and a
radionuclide.
21. The method of claim 17, further comprising isolating the Her2
binding protein of claim 1 from the host cell or from medium in
which the host cell is or was cultured.
22. The method of claim 12, wherein the disease or disorder
associated with Her2 expression is cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to new Her2 binding molecules
based on di-ubiquitin muteins. The invention further refers to Her2
binding proteins optionally fused or conjugated to a moiety
modulating pharmacokinetics or to a therapeutically or
diagnostically active component. The invention further relates to
the use of these Her2 binding proteins in medicine, preferably for
use in the diagnosis or treatment of cancer.
Background of the Invention
[0002] Increased expression of the membrane-bound receptor tyrosine
kinase Her2 plays an important role in the development and
progression of many breast carcinomas, but also in ovarian,
stomach, and uterine cancer, particularly with aggressive forms of
cancer. Overexpression of this oncogene is reported for
malignancies, predominantly in malignancies of epithelial origin,
and is associated with cancer recurrence and poor prognosis. The
three domain protein (extracellular, transmembrane, intracellular
tyrosine kinase domain) is mediating cell proliferation and
inhibiting apoptosis. Upon binding of a ligand to the extracellular
domain of Her2, Her2 forms dimers with the receptor whereby the
intracellular domain of Her2 is activated which mediates cellular
processes such as proliferation, differentiation, migration, or
apoptosis. Thus, modulating the function of Her2 is an important
approach for the development of cancer therapeutics, in particular
those based on monoclonal antibodies binding to the extracellular
domain of Her2. Therapeutic anti-Her2 monoclonal antibodies such as
Trastuzumab or Pertuzumab are available for treatments of cancer,
in particular breast cancer.
Technical Problems Underlying the Present Invention
[0003] However, monoclonal antibodies have major disadvantages such
as a complex molecular structure, a large size, and challenging
production methods. Furthermore, treatment of diseases with
currently available Her2 binding molecules is not effective in all
patients and may have severe side effects.
[0004] Needless to say that there is a strong medical need to
effectively treat cancer with improved novel agents, in particular
efficient tumor targeted therapeutics and diagnostics. There is an
ongoing need to find alternative to current therapies and
diagnosis, i.e. to substitute Her2 monoclonal antibodies by smaller
and less complex Her2 specific molecules such as non-immunoglobulin
based Her2 binding agents.
[0005] To overcome the disadvantages of antibodies, novel Her2
binding molecules suitable for diagnostic and therapeutic
applications should include characteristics such as affinity to
Her2, specificity to Her2, and high stability. It is thus an
objective of the present invention to provide novel Her2 binding
non-immunoglobulin molecules for new and improved strategies in the
treatment and diagnosis of cancer with Her2 overexpression. In
particular, it is an objective to provide novel binding proteins
which have high affinity and specificity to Her2, combined with a
less complex and smaller structure, for example for enabling a
simplified molecular engineering.
[0006] A solution is provided in this invention by small Her2
binding proteins such as non-immunoglobulin based binding agents,
in particular by Her2 binding molecules based on ubiquitin muteins
(also known as Affilin.RTM. molecules). Compared to antibodies, a
significant advantage of the Her2 binding proteins of the invention
is the reduced complexity in terms of (i) reduced size (e.g. of
maximal 152 amino acids), (ii) simple molecular structure (one
chain compared to four chains of an antibody), and (iii)
posttranslational modifications possible but not required for full
functionality. The binding proteins of the invention provide
molecular formats with favorable physicochemical properties (such
as stability and solubility), high-level expression, and allow easy
production methods. The Her2 specific Affilin molecules of the
invention are characterized by high affinity for Her2, by
specificity for a Her2, and by high stability, and provide novel
therapeutic and diagnostic possibilities.
[0007] The above-described objectives and advantages are achieved
by the subject-matters of the enclosed independent claims. The
present invention meets the needs presented above by providing
examples for specific Her2 binding proteins based on di-ubiquitin
muteins with substitutions in at least 12 amino acid positions of
di-ubiquitin. Preferred embodiments of the invention are included
in the dependent claims as well as in the following description,
examples and figures. The above overview does not necessarily
describe all problems solved by the present invention.
SUMMARY OF THE INVENTION
[0008] In a first aspect the present invention relates to a Her2
binding protein wherein the Her2 binding protein comprises an amino
acid sequence wherein at least 12 amino acids selected from
positions R42, 144, H68, V70, R72, L73, R74, K82, L84, Q138, K139,
E140, S141, and T142 of di-ubiquitin (SEQ ID NO: 4) are substituted
and wherein the Her2 binding protein has at least 85% sequence
identity to di-ubiquitin (SEQ ID NO: 4) and wherein the Her2
binding protein has a binding affinity (K.sub.D) of less than 700
nM for Her2, preferably the binding affinity determined by ELISA or
by surface plasmon resonance assays.
[0009] Another aspect of the present invention relates to a Her2
binding protein further comprising at least one additional
molecule, preferably selected from at least one member of the
groups (i), (ii) and (iii) consisting of (i) a moiety modulating
pharmacokinetic behavior selected for example from a polyethylene
glycol, a human serum albumin (HSA), a human serum albumin binding
protein, an albumin-binding peptide, or an immunoglobulin or
immunoglobulin fragments, a polysaccharide, and, (ii) a
therapeutically active component, optionally selected for example
from a monoclonal antibody or a fragment thereof, a cytokine, a
chemokine, a cytotoxic compound, an enzyme, or derivatives thereof,
or a radionuclide, and (iii) a diagnostic component, optionally
selected for example from a fluorescent compound, a
photosensitizer, a tag, an enzyme, or a radionuclide.
[0010] The present invention also provides, in further aspects, a
nucleic acid or nucleic acids encoding the Her2 binding proteins
comprising or consisting of a binding protein of the present
invention, as well as a vector or vectors comprising said nucleic
acid or nucleic acids, and a host cell or host cells comprising
said vector or vectors. Another aspect relates to said Her2 binding
protein for use in diagnostics or medicine, preferably for use in
the diagnosis or treatment of cancer, or a nucleic acid molecule
encoding said Her2 binding protein, or a vector comprising said
Her2 binding protein, or a host cell comprising said Her2 binding
protein, or a non-human host comprising said Her2 binding
protein.
[0011] Another aspect relates to a composition comprising the Her2
binding protein of the invention, the nucleic acid molecule of the
invention, the vector of the invention, or the host cell of the
invention, preferably for use in the diagnosis or treatment of
cancer.
[0012] Another aspect of the present invention relates to a method
for the production of a Her2 binding protein of any of the
preceding aspects of the invention comprising culturing of host
cells under suitable conditions and optionally isolation of the
Her2 binding protein produced.
[0013] This summary of the invention does not necessarily describe
all features of the present invention. Other embodiments will
become apparent from a review of the ensuing detailed
description.
BRIEF DESCRIPTION OF THE FIGURES
[0014] The Figures show:
[0015] FIG. 1 shows Her2 binding Affilin molecules.
[0016] FIG. 1A lists positions of di-ubiquitin (SEQ ID NO: 4) that
are substituted in order to generate a Her2 binding protein. In the
first row, the corresponding amino acid position is listed. All
Her2 binding proteins (for example, SEQ ID NOs: 5-38) are
substituted at least in 12 positions selected from positions R42,
144, H68, V70, R72, L73, R74, K82, L84, Q138, K139, E140, S141, and
T142 of SEQ ID NO: 4. A "." In the table refers to a wild type
position (unchanged); for example, as exemplified in SEQ ID Nos: 6,
31, 33, 34, 35, 36, and 37.
[0017] FIG. 1B shows the same amino acid exchanges as FIG. 1A,
however, the exchanges are translated according to the following
code which groups amino acids with similar biophysical properties.
A waved line "-" is the symbol for polar amino acids (T, S, N, or
Q) "H" is the symbol for hydrophobic amino acids (e.g. A, M, L, V,
I) "o" is the symbol for aromatic amino acids (e.g. F, W, Y),
"+"the symbol for basic amino acids (e.g. K, R, H), "-" the symbol
for acidic amino acids (e.g. D, E), and "G" corresponds to
Glycine.
[0018] FIG. 1C lists further substitutions; all Her2 binding
proteins have 0, 1, 2, 3, 4, 5, or 6 further modifications in
addition to the atleast 12 substitutions selected from amino acid
positions R42, I44, H68, V70, R72, L73, R74, K82, L84, Q138, K139,
E140, S141, and T142 of SEQ ID NO: 4.
[0019] FIG. 1D shows the same amino acid exchanges as FIG. 1C,
however, the exchanges are translated according to the code which
groups amino acids with similar biophysical properties as described
in FIG. 1B.
[0020] FIG. 2. Biochemical characterization of Her2 binding Affilin
molecules (for example, SEQ ID NOs: 5-38). Shown are binding
affinities (K.sub.D) as obtained from SPR assay (Biacore; third
column of the table) and temperature stability (DSF; fourth column
of the table).
[0021] FIG. 3. Analysis of Her2 binding proteins via label-free
interaction assays using SPR (Biacore). Different concentrations of
Affilin proteins (0, 0.137, 0.4115, 1.2345, 3.7037, 11.11, and
33.33 nM) were analyzed for binding to Her2 immobilized on a chip
(Biacore) to analyze the interaction between the Affilin protein
and Her2. FIG. 3A shows the binding kinetics of Affilin-142628 to
Her2. FIG. 3B shows the binding kinetics of Affilin-144633 to
Her2.
[0022] FIG. 4. Functional characterization of Her2 binding proteins
confirming binding to cellular Her2. The figure shows binding to
exogenously Her2 expressing SkBr3 cells as determined by FACS
analysis. Her2 binding proteins ("Affilin") show binding at 50 nM
on SkBr3 cells (dark grey bars) and no activity on HEK/293 cells
(see FIG. 4A and FIG. 4B). Weak or no binding to Her2 expressing
SkBr3 was observed for Affilin-142655 (referred to as "142655"),
Affilin-141965, Affilin-142465 (referred to as "142465"),
Affilin-142502 (referred to as "142502"), and di-ubiquitin
(referred to as "di-ubi").
[0023] FIG. 5. Concentration dependent functional binding of Her2
binding proteins to exogenously Her2 expressing SkBr3 cells as
determined by flow cytometry analysis. Shown is a dilution series
of 333 nM to 5.6 pM of binding protein. Affilin-141926 (FIG. 5 A)
and Affilin-141890 (FIG. 5 B) show a concentration depending
binding on SkBr3-cells. FIG. 6. Functional characterization of Her2
binding proteins confirming binding to exogenously Her2
overexpressing CHO-K1 cells as determined by flow cytometry
analysis. Her2 binding proteins show binding on CHO-K1-Her2 cells
at concentrations of 50 nM, 5 nM, and 0.5 nM. FIG. 6A shows the
Her2-binding of Affilin-142627, Affilin-142628, Affilin-142654, and
Affilin-141884; FIG. 6B shows cellular Her 2 binding of
Affilin-144631, Affilin-144632, Affilin-144633, Affilin-144634,
Affilin-144635, Affilin-144636, Affilin-144637, and FIG. 6 C shows
cellular Her 2 binding of Affilin-144567, and only low levels of
binding of 142502 at 500 nM. Thus, cellular Her2 binding was
confirmed for all binding molecules except 142502 even at the
lowest concentration tested. Di-ubiquitin showed no binding on
CHO-K1-Her2-cells (shown, for example, in FIG. 6 B).
[0024] FIG. 7. Concentration dependent binding of Affilin-142628.
The figure shows binding of Affilin-142628 to exogenously Her2
expressing CHO-K1 cells as determined by flow cytometry (FACS
analysis)(control: empty vector CHO-K1-pEntry cells). Histograms at
different Affilin protein concentrations of 50 nM, 5 nM and 0.5 nM
are shown in comparison to di-ubiquitin 139090 (SEQ ID NO: 4).
Affilin-142628 induces a concentration depending shift on the
Her2-overexpressing cell line.
[0025] FIG. 8. Concentration dependent functional binding of Her2
binding proteins to exogenously Her2 expressing SkBr3-cells as
determined by flow cytometry. A dilution series of 100 nM to 0.06
pM of Affilin-142628 was used to analyze the interaction with Her2
overexpressing SkBr3 cells. FIG. 8 shows a concentration dependent
binding of Affilin-142628.
[0026] FIG. 9 shows the binding analysis of different Her2 binding
proteins on Her2-overexpressing SkBr3-cells by immunofluorescence
staining. FIG. 9A shows concentrations of 50 nM Affilin-141884,
Affilin-142628, Affilin-141926, Affilin-144637, Affilin-142418.
FIG. 9B shows concentrations of 50 nM Affilin-144567, di-ubiquitin
(139090), PBS, and Trastuzumab (Herceptin). Affilin-141884,
Affilin-142628, Affilin-141926, Affilin-144637 and Affilin-142418
show a strong binding on the Her2-overexpressing cell line, whereas
Affilin-144567 and di-ubiquitin (139090) do not bind to Her2 on
SkBr-3 cells.
[0027] FIG. 10 confirms that Her2 binding proteins bind to SKOV-3
xenograft tumor tissue. Shown is an immunohistological staining of
50 nM Affilin-141884 and 50 nM Affilin-142628 on Her2-expressing
tumor tissue derived from human ovarian adenocarcinoma cells.
Affilin-141884 and Affilin-142628 show a strong binding on
Her2-expressing tissue. Di-ubiquitin (139090) shows no binding on
SKOV-3 tissue slides.
[0028] FIG. 11 shows an immunohistological binding analysis of Her2
binding proteins on Her2-expressing SKOV-3-tumor tissue slides and
lung tissue slides without Her2 expression. Affilin-141884 and
Affilin-142628 show strong binding at 20 nM on SKOV-3 tissue. No
binding to lung tissue was observed. In addition, no binding of
Affilin-141884 and Affilin-142628 to tissue obtained from liver,
heart muscle, and ovary was observed.
[0029] FIG. 12 shows that Affilin-142628 and Affilin-143692 bind to
different Her2 epitopes (competition analysis; binding analysis
SPR). These Her2 binding proteins do not compete for Her2 binding
and thus, use different or non-overlapping epitopes of Her2.
[0030] FIG. 13 shows that the Her2 binding proteins Affilin-142628
and Affilin-143692 bind to different Her2 epitopes than Trastuzumab
(Herceptin).
[0031] FIG. 14 shows the simultaneous binding of a bispecific
fusion protein to Her2 and EGFR. The fusion of a Her2 binding
protein to an EGFR specific monoclonal antibody (Cetuximab) enables
bispecific targeting, as shown for example for fusion proteins SEQ
ID NOs: 44-47.
[0032] FIG. 15 shows the flow cytometric binding analysis of a
bispecific fusion protein comprising an Her2 specific Affilin fused
to the C-terminus of the light chain of Cetuximab (CL-141926; SEQ
ID NO: 44) on Her2 overexpressing CHO K1 cells (FIG. 15B) and on
EGFR overexpressing CHO K1 cells (FIG. 15B). The fusion protein
shows binding to both extracellular targets. The figure shows the
median fluorescence intensity (MFI), representing the binding of
the Affilin-antibody fusion protein to EGFR and to Her2 expressing
cells at the indicated concentrations.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Before the present invention is described in more detail
below, it is to be understood that this invention is not limited to
the particular methodology, protocols and reagents described herein
as these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art to which this invention belongs.
[0034] Preferably, the terms used herein are defined as described
in "A multilingual glossary of biotechnological terms: (IUPAC
Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds.
(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
[0035] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variants such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps. Several documents (for example:
patents, patent applications, scientific publications,
manufacturers specifications, instructions, GenBank Accession
Number sequence submissions etc.) are cited throughout the text of
this application. Nothing herein is to be construed as an admission
that the invention is not entitled to antedate such disclosure by
virtue of prior invention. Some of the documents cited herein are
characterized as being "Incorporated by reference". In the event of
a conflict between the definitions or teachings of such
incorporated references and definitions or teachings recited in the
present specification, the text of the present specification takes
precedence. All sequences referred to herein are disclosed in the
attached sequence listing that, with its whole content and
disclosure, is a part of this specification.
[0036] General Definitions of Important Terms Used in the
Application
[0037] The terms "protein" and "polypeptide" refer to any chain of
two or more amino acids linked by peptide bonds, and does not refer
to a specific length of the product. Thus, "peptides", "protein",
"amino acid chain," or any other term used to refer to a chain of
two or more amino acids, are included within the definition of
"polypeptide," and the term "polypeptide" may be used instead of,
or interchangeably with any of these terms. The term "polypeptide"
is also intended to refer to the products of post-translational
modifications of the polypeptide, including without limitation
glycosylation, acetylation, phosphorylation, amidation, proteolytic
cleavage, modification by non-naturally occurring amino acids and
similar modifications which are well known in the art. Thus,
binding proteins comprising two or more protein moieties also fall
under the definition of the term "protein" or "polypeptides".
[0038] The term "ubiquitin" or "unmodified ubiquitin" refers to
ubiquitin in accordance with SEQ ID NO: 1 and to proteins with at
least 95% identity, such as SEQ ID NO: 2 (point mutations in
positions 45, 75, 76 which do not influence binding to a target),
to a di-ubiquitin according to SED ID NO: 4 and to proteins with at
least 95% identity, such as , di-ubiquitin according to SEQ ID NO:
48, and according to the following definition. Particularly
preferred are ubiquitin molecules from mammals, e.g. humans,
primates, pigs, and rodents. On the other hand, the ubiquitin
origin is not relevant since according to the art all eukaryotic
ubiquitins are highly conserved and the mammalian ubiquitins
examined up to now are even identical with respect to their amino
acid sequence. In addition, ubiquitin from any other eukaryotic
source can be used. For instance ubiquitin of yeast differs only in
three amino acids from the wild-type human ubiquitin (SEQ ID NO:
1).
[0039] The term "di-ubiquitin" refers to a protein comprising two
unmodified ubiquitin moieties linked to each other in head-to-tail
orientation. An example is given in SED ID NO: 4 (point mutations
in positions 45, 75, 76, 151, 152 of wildtype ubiquitin; these
point mutations do not influence binding to a target; clone
139090), and in SEQ ID NO: 48. The amino acid sequence identity
between SEQ ID NO: 4 and SEQ ID NO: 48 is 96.7%. A di-ubiquitin
according to the present invention is an artificial protein of 152
amino acids consisting of two ubiquitin moieties directly linked to
each other without a peptide linker between the two ubiquitin
moieties. A di-ubiquitin as understood herein is a protein with at
least 95% identity to SEQ ID NO: 4.
[0040] The terms "modified ubiquitin" and "ubiquitin mutein" and
"Affilin" are all used synonymously and can be exchanged. The term
"modified ubiquitin" or "ubiquitin mutein" or "Affilin" as used
herein refers to derivatives of ubiquitin which differ from said
unmodified ubiquitin by amino acid exchanges, insertions, deletions
or any combination thereof, provided that the ubiquitin mutein has
a specific binding affinity to a target epitope or antigen which is
at least 10fold lower or absent in unmodified ubiquitin. This
functional property of an ubiquitin mutein (Affilin; modified
ubiquitin) is a de novo created function.
[0041] The term "Affilin.RTM." (registered trademark of Scil
Proteins GmbH) refers to non-immunoglobulin derived binding
proteins based on ubiquitin muteins. An Affilin protein is not a
natural ubiquitin existing in or isolated from nature, for example,
as shown in SEQ ID NO: 1. The scope of the invention excludes
unmodified ubiquitin. An Affilin molecule according to this
invention comprises, essentially consists, or consists of either
two differently modified ubiquitin moieties linked together in a
head-to-tail fusion or an Affilin molecule that comprises,
essentially consists, or consists of one modified ubiquitin moiety.
A "head-to-tail fusion" is to be understood as fusing two proteins
together by connecting them in the direction (head) N--C--N--C--
(tail) (tandem molecule), as described for example in EP2379581B1
which is incorporated herein by reference. The head part is
designated as the first moiety and the tail part as the second
moiety. In this head-to-tail fusion, two moieties may be connected
directly without any linker (e.g. SEQ ID NOs: 5-38). Alternatively,
the fusion of two proteins can be performed via linkers, for
example, a polypeptide linker, as described herein.
[0042] The term "substitution" includes "conservative" and
"non-conservative" substitutions. "Conservative substitutions" may
be made, for instance, on the basis of similarity in polarity,
charge, size, solubility, hydrophobicity, hydrophilicity, and/or
the amphipathic nature of the amino acid residues involved. Amino
acids can be grouped into the following standard amino acid groups:
(1) hydrophobic side chains: Ala (A), Met (M), Leu (L), Val (V),
Ile (I); (symbol "H" in FIG. 1) (2) acidic polar side chain: Asp
(D), Glu (E) (symbol "-" in FIG. 1); (3) basic side chain polarity:
Lys (K), Arg (R), His (H) (symbol "+" in FIG. 1); (4) aromatic
amino acids: Trp (W), Tyr (Y), Phe (F) (symbol "o" in FIG. 1); (5)
polar amino acids: Thr (T), Ser (S), Asn (N), Gln (Q) (symbol
"wave" in FIG. 1); (6) residues that influence chain orientation:
Gly (G), Pro (P); and (7) Cys (C). As used herein, "conservative
substitutions" are defined as exchanges of an amino acid by another
amino acid listed within the same group of the standard amino acid
groups shown above. For example, the exchange of Asp by Glu retains
one negative charge in the so modified polypeptide. In addition,
Gly and Pro may be substituted for one another based on their
ability to disrupt .alpha.-helices. Some preferred conservative
substitutions within the above groups are exchanges within the
following sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr;
(ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the
known genetic code, and recombinant and synthetic DNA techniques,
the skilled scientist can readily construct DNAs encoding the
conservative amino acid variants. As used herein, "non-conservative
substitutions" or "non-conservative amino acid exchanges" are
defined as exchanges of an amino acid by another amino acid listed
in a different group of the amino acid groups (1) to (7) shown
above.
[0043] The term "insertions" comprises the addition of amino acids
to the original amino acid sequence of ubiquitin wherein the
ubiquitin remains stable without significant structural change.
Naturally, loop regions connect regular secondary structure
elements. The structure of human unmodified ubiquitin (SEQ ID NO:
1) reveals six loops at amino acid regions 8-11, 17-22, 35-40,
45-47, and 50-63 which connect secondary structure elements such as
beta sheets and alpha helix. In one embodiment of the invention,
Her2 binding proteins are disclosed comprising a ubiquitin mutein
having a combination of an insertion and substitutions. In one
embodiment, ubiquitin muteins have insertions of 2-10 amino acid
residues, preferably within the most N-terminal loop within amino
acids 8-11. Specifically, the number of amino acid residues to be
inserted is 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2-10 amino acid
residues, most preferred 6-9 amino acid residues.
[0044] The term "antibody" as used in accordance with the present
invention comprises monoclonal antibodies having two heavy chains
and two light chains (immunoglobulin or IgG antibodies).
Furthermore, also fragments or derivatives thereof, which still
retain the binding specificity, are comprised in the term
"antibody". The term "antibody" also includes embodiments such as
chimeric (human constant domain, non-human variable domain), single
chain and humanized (human antibody with the exception of non-human
CDRs) antibodies. Full-length IgG antibodies consisting of two
heavy chains and two light chains are most preferred in this
invention. Heavy and light chains are connected via non-covalent
interactions and disulfide bonds.
[0045] In the present specification, the terms "target antigen",
"target", "antigen" and "binding partner" are all used synonymously
and can be exchanged. Preferably the target is one of the targets
defined herein below. The term "antigen", as used herein, is to be
interpreted in a broad sense and includes any target moiety that is
bound by the binding moieties of the binding proteins of the
present invention.
[0046] The terms "protein capable of binding" or "binding protein"
or "binding Her2" or "binding affinity for" according to this
invention refer to a protein comprising a binding capability to a
defined target antigen. The term "Her2 binding protein" refers to a
protein with high affinity binding capability to Her2.
[0047] An "antigen binding site" refers to the site, i.e. one or
more amino acid residues, of an antigen binding molecule which
provide interaction with the antigen. A native immunoglobulin
molecule typically has two antigen binding sites, a Fab molecule
typically has a single antigen binding site.
[0048] The term "epitope" includes any molecular determinant
capable of being bound by an antigen binding protein as defined
herein and is a region of a target antigen that is bound by an
antigen binding protein that targets that antigen, and when the
antigen is a protein, it may include specific amino acids that
directly contact the antigen binding protein. In a conformational
epitope, amino acid residues are separated in the primary sequence,
but are located near each other on the surface of the molecule when
the polypeptide folds into the native three-dimensional structure.
A linear epitope is characterized by two or more amino acid
residues which are located adjacent in a single linear segment of a
protein chain. In other cases, the epitope may include determinants
from posttranslational modifications of the target protein such as
glycosylation, phosphorylation, sulfatation, acetylation, fatty
acids or others.
[0049] The term "fused" means that the components (e.g. an Affilin
molecule and a monoclonal antibody or a Fab fragment) are linked by
peptide bonds, either directly or via peptide linkers.
[0050] The term "fusion protein" relates to a protein comprising at
least a first protein joined genetically to at least a second
protein. A fusion protein is created through joining of two or more
genes that originally coded for separate proteins. Thus, a fusion
protein may comprise a multimer of different or identical binding
proteins which are expressed as a single, linear polypeptide. It
may comprise one, two, three or even more first and/or second
binding proteins. A fusion protein as used herein comprises at
least a first binding protein (e.g. Affilin) which is fused with at
least a second binding protein, e.g. a monoclonal antibody or a
fragment thereof. Such fusion proteins may further comprise
additional domains that are not involved in binding of the target,
such as but not limited to, for example, multimerization moieties,
polypeptide tags, polypeptide linkers.
[0051] The term "conjugate" as used herein relates to a protein
comprising or essentially consisting of at least a first protein
attached chemically to other substances such as to a second protein
or a non-proteinaceous moiety. The conjugation can be performed by
means of organic synthesis or by use of enzymes including natural
processes of enzymatic post-translational modifications. Examples
for protein conjugates are glycoproteins (conjugated protein with
carbohydrate component) or lipoproteins (conjugated protein with
lipid component). The molecule can be attached e.g. at one or
several sites through any form of a linker. Chemical coupling can
be performed by chemistry well known to someone skilled in the art,
including substitution (e.g. N-succinimidyl chemistry), addition or
cycloaddition (e.g. maleimide chemistry or click chemistry) or
oxidation chemistry (e.g. disulfide formation). Some examples of
non-proteinaceous polymer molecules which are chemically attached
to protein of the invention are hydroxyethyl starch, polyethylene
glycol, polypropylene glycol, dendritic polymers, or
polyoxyalkylene and others.
[0052] A fusion protein or protein conjugate may further comprise
one or more reactive groups or peptidic or non-peptidic moieties
such as ligands or therapeutically or diagnostically relevant
molecules such as radionuclides or toxins. It may also comprise
small organic or non-amino acid based compounds, e.g. a sugar,
oligo- or polysaccharide, fatty acid, etc. Methods for attaching a
protein of interest to such non-proteinaceous components are well
known in the art, and are thus not described in further detail
here.
[0053] The terms "bispecific binding molecule" or "multispecific
binding molecule" mean that the antigen binding molecule is able to
specifically bind two or multiple different epitopes. Typically, a
bispecific antigen binding molecule comprises two antigen binding
sites, each of which is specific fora different epitope. In certain
embodiments the bispecific antigen binding molecule is capable of
simultaneously binding two epitopes, particularly two epitopes
expressed on two distinct cells. The term "bispecific binding
molecule" or "bispecific binding protein" means that binding
proteins of the present invention are capable of specifically
binding to two different epitopes. Moreover, the bispecific binding
molecule of the present invention is capable of binding to two
different epitopes at the same time. This means that a bispecific
construct is capable of simultaneously binding to at least one
epitope "A" and at least one epitope "B", wherein A and B are not
the same. The two epitopes may be located on the same or different
target antigens which means that the fusion molecules of the
present invention can bind one target at two different epitopes or
two target antigens each with its own epitope. Similarly,
"multispecific binding molecules" are capable of binding multiple
epitopes at the same time wherein the epitopes may be located on
the same or different antigens.
[0054] Alternatively, said binding proteins may bind to different,
non-overlapping epitopes on the same or different target molecules
and are thus classified as bispecific, trispecific, multispecific,
etc., for example .alpha..beta., .beta..gamma., .alpha..delta.,
.alpha..beta..gamma., .alpha..beta..gamma..delta. binding to
epitopes AB, BC, AD, ABC or ABCD, respectively. For example, fusion
proteins with Her2-specific Affilin and anti-EGFR-monoclonal
antibody are bispecific.
[0055] The term "multimeric binding molecules" refers to fusion
proteins that are multivalent and/or multispecific, comprising two
or more moieties (i.e. bivalent or multivalent) of binding protein
.alpha., .beta. and/or .gamma. etc., e.g. .alpha..alpha.,
.beta..beta..beta., .alpha..alpha..beta.,
.alpha..alpha..beta..beta., .alpha..gamma..gamma.,
.beta..beta..gamma., .alpha..beta..gamma..delta..delta., etc. For
example, aaf3y is trispecific and bivalent with respect to epitope
A. For example, the fusion proteins of Her2-specific Affilin and
monoclonal antibodies as described herein are at least "bivalent"
because they comprise at least two binding proteins (for example,
an Affilin and an monoclonal antibody). Said binding proteins may
bind specifically to the same or overlapping epitopes on a target
antigen (monospecific), e.g. the composition of the binding protein
may be described by (.alpha.).sub.2, (.alpha.).sub.3,
(.alpha.).sub.4, (.beta.).sub.2, (.beta.).sub.3, (.beta.).sub.4
etc. In this case, the fusion molecules are monospecific but
bivalent, trivalent, tetravalent, or multivalent for the epitope A
or epitope B, respectively.
[0056] The term "amino acid sequence identity" refers to a
quantitative comparison of the identity (or differences) of the
amino acid sequences of two or more proteins. "Percent (%) amino
acid sequence identity" with respect to a reference polypeptide
sequence is defined as the percentage of amino acid residues in a
sequence that are identical with the amino acid residues in the
reference polypeptide sequence, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity.
[0057] To determine the sequence identity, the sequence of a query
protein is aligned to the sequence of a reference protein, for
example, to SEQ ID NO: 4 (di-ubiquitin) or to SEQ ID NO: 1
(ubiquitin). Methods for alignment are well known in the art. For
example, for determining the extent of an amino acid sequence
identity of an arbitrary polypeptide relative to the amino acid
sequence of SEQ ID NO: 4 or SEQ ID NO: 1, the SIM Local similarity
program is preferably employed (Xiaoquin Huang and Webb Miller
(1991), Advances in Applied Mathematics, vol. 12: 337-357), that is
freely available (see also:
http://www.expasy.org/tools/sim-prot.html). For multiple alignment
analysis ClustalW is preferably used (Thompson et al. (1994)
Nucleic Acids Res., 22(22): 4673-4680).
[0058] In the context of the present invention, the extent of
sequence identity between a modified sequence and the sequence from
which it is derived (also termed "parental sequence") is generally
calculated with respect to the total length of the unmodified
sequence, if not explicitly stated otherwise. Each amino acid of
the query sequence that differs from the reference amino acid
sequence at a given position is counted as one difference. An
insertion or deletion in the query sequence is also counted as one
difference. For example, an insertion of a linker between two
ubiquitin moieties is counted as one difference compared to the
reference sequence. The sum of differences is then related to the
length of the reference sequence to yield a percentage of
non-identity. The quantitative percentage of identity is calculated
as 100 minus the percentage of non-identity. In specific cases of
determining the identity of ubiquitin muteins aligned against
unmodified ubiquitin, differences in positions 45, 75 and/or 76 are
not counted, in particular, because they are not relevant for the
novel binding capability of the ubiquitin mutein. The ubiquitin
moiety can be modified in amino acid residues 45, 75 and/or 76
without affecting its binding capability; said modifications might,
however, be relevant for achieving modifications in the biochemical
properties of the mutein. Generally, a ubiquitin used as starting
material for the modifications has an amino acid identity of at
least 95%, of at least 96% or of at least 97%, or of at least an
amino acid sequence identity of 98% to SEQ ID NO: 1. Thus, a
polypeptide which is, for example, 95% "identical" to a reference
sequence may comprise, for example, five point mutations or four
point mutations and one insertion etc., per 100 amino acids,
compared to the reference sequence.
[0059] The term "dissociation constant" or "K.sub.D" defines the
specific binding affinity. As used herein, the term "K.sub.D"
(usually measured in "mol/L", sometimes abbreviated as "M") is
intended to refer to the dissociation equilibrium constant of the
particular interaction between a first compound and a second
compound. In the context of the present invention, the term K.sub.D
is particularly used to describe the binding affinity between a
Her2-binding protein and Her2. A high affinity corresponds to a low
value of K.sub.D. Thus, the expression "a K.sub.D of at least e.g.
10.sup.-7 M" means a value of 10.sup.-7M or lower (binding more
tightly). 1.times.10.sup.-7M corresponds to 100 nM. A value of
10.sup.-5 M and below down to 10.sup.-12 M can be considered as a
quantifiable binding affinity. Depending on the application a value
of 10.sup.-7 to 10.sup.-12 M is preferred for chromatographic
applications or for diagnostic or therapeutic applications. In
accordance with the invention the affinity for the target binding
is in the range of 7.times.10.sup.-7M (700 nM) or less. Final
target binding affinity can be ideally 10.sup.-9M (1 nM) or
less.
[0060] Binding proteins of the invention comprise two ubiquitin
muteins linked directly without any linker to result in unique and
high affinity Her2 binding proteins with substitutions at least in
12, 13, or 14 positions selected from 42, 44, 68, 70, 72, 73, 74,
82, 84, 138, 139, 140, 141, and 142 of di-ubiquitin (SEQ ID NO: 4
or SEQ ID NO: 48), and optionally in 0, 1, 2, 3, 4, 5, or 6 further
substitutions.
[0061] Binding proteins of the invention can be fused, e.g.
genetically, to other functional protein moieties. In the context
of such fusion proteins of the invention the term "linker" refers
to a single amino acid or a polypeptide that joins at least two
other protein molecules covalently. The linker is e.g. genetically
fused to the first and second protein or protein moieties to
generate a single, linear polypeptide chain. The length and
composition of a linker may vary between at least one and up to
about 50 amino acids. Preferably, the linker length is between one
and 30 amino acids. More preferably, the peptide linker has a
length of between 1 and 20 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. It
is preferred that the amino acid sequence of the peptide linker is
not immunogenic to human beings, stable against proteases and
optionally does not form a secondary structure. An example is a
linker comprised of small amino acids such as glycine or serine.
The linkers can be glycine-rich (e.g., more than 50% of the
residues in the linker can be glycine residues). Preferred are
glycine-serine-linkers of variable length consisting of glycine and
serine residues only. In general, linkers of the structure
(SGGG).sub.n or permutations of SGGG, e.g. (GGGS).sub.n, can be
used wherein n can be any number between 1 and 6, preferably 1 or 2
or 3. Also preferred are linkers comprising further amino acids.
Other linkers for the genetic fusion of proteins are known in the
art and can be used. In one embodiment of the invention, the first
binding protein (e.g. Affilin) and the second binding protein (e.g.
monoclonal antibody or fragment thereof) are linked via a
(G.sub.3S).sub.4 linker.
[0062] In case of chemical conjugates of the binding proteins of
the invention, the term "linker" refers to any chemical moiety
which connects the Her2 binding protein with other proteinaceous or
non-proteinaceous moieties either covalently or non-covalently,
e.g., through hydrogen bonds, ionic or van der Waals interactions,
such as two complementary nucleic acid molecules attached to two
different moieties that hybridize to each other, or chemical
polymers such as polyethylene glycol or others. Such linkers may
comprise reactive groups which enable chemical attachment to the
protein through amino acid side chains, the N-terminal
.alpha.-amino- or C-terminal carboxy-group of the protein. Such
linkers and reactive groups are well-known to those skilled in the
art and not described further.
[0063] Her2 (Human Epidermal Growth Factor Receptor 2; synonym
names are ErbB-2, Neu, CD340 or p185) is a 185-kDa receptor first
described in 1984 (Schlechter et al (1984) Nature 312:513-516).
Amplification or over-expression of this gene has been shown to
play an important role in the pathogenesis and progression of
certain aggressive types of breast cancer, and Her2 is known as an
important biomarker and target of therapy for the disease. Other
tumors where Her2 plays a role include ovarian cancer and gastric
cancer. Human Her2 is represented by the NCBI accession number
NP_004439; the extracellular domain (residues 1-652) of Her2 is
represented by the uniprot Accession Number p04626. The term "Her2"
comprises all polypeptides which show a sequence identity of at
least 70%, 80%, 85%, 90%, 95%, 96% or 97% or more, or 100% to
NP_004439 and have the functionality of Her2.
DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION
[0064] The Her2 binding protein of the invention comprises,
essentially consists of or consists of two differently modified
ubiquitin moieties directly connected without a linker in
head-to-tail orientation. The Her2 binding protein of the invention
has an amino acid identity of at least 85% to di-ubiquitin (SEQ ID
NO: 4); i.e. a maximum of 23 amino acids are modified in
di-ubiquitin (SEQ ID NO: 4) (152 amino acids total) to generate a
novel binding property of di-ubiquitin (SEQ ID NO: 4) to Her2.
Further preferred amino acid identities of the novel Her2 binding
proteins are at least 86%, at least 87% (corresponding to 20 amino
acids modified), at least 88%, or at least 89%, at least 90%
(corresponding to 15 amino acids modified), at least 91%
(corresponding to 14 amino acids modified), at least 92%
(corresponding to 12 amino acids modified) to di-ubiquitin (SEQ ID
NO: 4). Thus, Her2 binding protein of the invention show 85% to 92%
identity to di-ubiquitin, more preferably between 87% to 91%
identity to di-ubiquitin. The Her2 binding protein of the invention
with binding affinity (K.sub.D) of less than 700 nM for Her2
comprises, essentially consists, or consists of an amino acid
sequence according to di-ubiquitin (SEQ ID NO: 4) wherein amino
acids selected from at least 12, 13, or 14 amino acids selected
from positions R42, I44, H68, V70, R72, L73, R74, K82, L84, Q138,
K139, E140, S141, and T142 of di-ubiquitin (SEQ ID NO: 4) are
substituted wherein the Her2 binding protein has at least 85%
sequence identity to di-ubiquitin (SEQ ID NO: 4). The Her2 binding
proteins as described in this invention show not more than 92%
sequence identity to SEQ ID NO: 4. The preferred Her2 binding
proteins comprise 152 amino acids with at least 85% to di-ubiquitin
(SEQ ID NO: 4), provided that at least 12, 13, or 14 amino acids
selected from positions R42, I44, H68, V70, R72, L73, R74, K82,
L84, Q138, K139, E140, S141, and T142 are substituted. All Her2
binding proteins have substitutions in positions R42, V70, R72,
L73, K82, L84, Q138, K139, E140, and T142, and preferably in
positions 144, H68, R74, and S141. Surprisingly, the specific
combination of substitutions in said 12, 13, or 14 positions of SEQ
ID NO: 4 results in high affinity Her2 binding proteins. These
proteins are artificial proteins that are created de novo. The Her2
binding proteins of the invention do not exist in nature. Examples
for de novo created Her2 binding proteins are provided in SEQ ID
NOs: 5-38. The Her2 binding protein is substituted in at least 12
positions selected from positions 42, 44, 68, 70, 72, 73, 74, 82,
84, 138, 139, 140, 141, and 142 of di-ubiquitin (SEQ ID NO: 4) and
has no further substitution, for example, SEQ ID NOs: 29, 33, one
additional substitution, for example, SEQ ID NOs: 27, 28, 31, 32,
two additional substitutions, for example, SEQ ID NOs: 14, 16, 21,
25, 26, 30, 35, three additional substitutions, for example, SEQ ID
NOs: 6, 12, 13, 15, 17, 18, 20, 34, 36, four additional
substitutions, for example, SEQ ID NOs: 10, 11, 19, 22, 23, 24,
five additional substitutions, for example, SEQ ID NOs: 5, 7, 8, 9,
or six additional substitutions, for example, SEQ ID NO: 37. For
example, further 1, 2, 3, 4, 5, or 6 substitutions in addition to
the at least 12 substitutions in positions 42, 44, 68, 70, 72, 73,
74, 82, 84, 138, 139, 140, 141, and 142 of SEQ ID NO: 4 may be
preferably selected from positions 6, 10, 11, 15, 20, 21, 23, 27,
28, 31, 34, 36, 40, 46, 48, 49, 52, 58, 62, 63, 75, 78, 88, 92, 95,
96, 98, 114, 120, 124, 131, 133, 144, and/or 147 of SEQ ID NO: 4
(see FIG. 1 and Table 1).
TABLE-US-00001 TABLE 1 Her2 binding proteins of the invention -
number of substitutions and degree of identity to SEQ ID NO: 4
Number of substitutions Number of total % SEQ in positions 42, 44,
68, additional number of identity ID 70, 72, 73, 74, 82, 84,
substi- substi- to SEQ NO: 138, 139, 140, 141, and 142 tutions
tutions ID NO: 4 5 14 6 20 86.8 7 14 6 20 86.8 8 14 6 20 86.8 9 14
6 20 86.8 10 14 5 19 87.5 11 14 5 19 87.5 19 14 5 19 87.5 22 14 5
19 87.5 23 14 5 19 87.5 24 14 5 19 87.5 12 14 4 18 88.2 13 14 4 18
88.2 15 14 4 18 88.2 17 14 4 18 88.2 18 14 4 18 88.2 20 14 4 18
88.2 14 14 3 17 88.9 16 14 3 17 88.9 21 14 3 17 88.9 25 14 3 17
88.9 26 14 3 17 88.9 30 14 3 17 88.9 25 14 3 17 88.9 6 13 4 17 88.9
36 13 4 17 88.9 27 14 2 16 89.5 28 14 2 16 89.5 32 14 2 16 89.5 34
12 4 16 89.5 29 14 1 15 90.3 31 13 2 15 90.3 33 12 1 13 91.4
[0065] Many examples of Her2 binding proteins are provided in this
invention (see, for example, FIG. 1a, SEQ ID NOs: 5-38). The Her2
binding Affilin molecules of the invention bind to the isolated
extracellular domain of Her2 with measurable binding affinity of
less than 700 nM, less than 500 nM, less than 100 nM, less than 20
nM, less than 10 nM (for example, SEQ ID NOs: 6, 14, 15, 18, 22,
24, 25, 26, 28, 35, 36, 38), and more preferred less than 1 nM (for
example, SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 16, 17, 19, 20,
23)(binding affinity as determined by Biacore; see, for example,
FIG. 2). The di-ubiquitin (SEQ ID NO: 4) does not naturally bind to
Her2 with any measurable binding affinity. All Her2 binding
proteins of the invention show de novo created binding to Her2 with
high affinity. Preferred substitutions of the Her2 binding protein
based on di-ubiquitin (SEQ ID NO: 4) are substitutions of amino
acids selected from position 70 and 140 by aromatic amino acids.
Further preferred substitutions of the Her2 binding protein based
on di-ubiquitin (SEQ ID NO: 4) are substitutions of amino acids
selected from position 42 by a polar amino acid, position 44 is
substituted by a hydrophobic or polar amino acid, position 68 is
substituted by an aromatic amino acid, position 72 is substituted
by a polar or aromatic amino acid, position 73 is substituted by
any amino acid but not basic or acidic amino acid, position 74 is
substituted by an aromatic, basic or polar amino acid, position 82
is substituted by any amino acid but not basic or acidic amino
acid, position 84 is substituted by a basic or acidic amino acid,
position 138 is substituted by a basic or acidic or polar amino
acid, position 139 is substituted by acidic or hydrophobic amino
acid or Glycine, position 141 is substituted by hydrophobic or
polar or basic amino acid, and/or position 142 is substituted by a
hydrophobic or polar amino acid. Preferred substitutions of the
Her2 binding protein based on di-ubiquitin (SEQ ID NO: 4) are
selected from R42T, R425, R42L, I44A, I44V, I44S, I44T, H68W, H68Y,
H68F, V70Y, V70W, R72T, R72F, R72G, R72Y, L73W, L735, L73V, L731,
R74Y, R74S, R74N, R74K, K82T, K82L, K82N, K821, K82Y, L84H, L84D,
L84E, L845, Q1385, Q138R, Q138E, K139E, K139G, K139L, E140W, S141A,
S141R, T1421, T142L, and/or T142N. Further preferred are Her2
binding proteins with a specific combination of amino acid
substitutions in SEQ ID NO: 4, for example, at least R42T, I44A,
H68W, V70Y, R72T, L73W, R74Y, K82T, L84H, as for example, in SEQ ID
NOs: 7-29 and 38.
[0066] Other preferred Her2 binding proteins with a specific
combination of amino acid substitutions in di-ubiquitin (SEQ ID NO:
4), are for example at least R42S, I44V, H68Y, V70Y, R72F, L735,
K82L, L84D, as for example, in SEQ ID NOs: 34, 35, 36, and 37.
Further preferred are Her2 binding proteins with a specific
combination of amino acid substitutions in di-ubiquitin (SEQ ID NO:
4), for example, Q138S, K139E, E140W, S141A, T142I (for example, in
SEQ ID NOs: 5, 7-29, 33, 36, 37), or Q138R, K139G, E140W, T142L
(for example, in SEQ ID NOs: 6, 34, 35), or Q138E, K139L, E140W,
S141R, T142N (for example, in SEQ ID NOs: 30, 31, 32).
[0067] Her2 binding proteins of the invention comprise amino acid
sequences selected from the group consisting of SEQ ID NO: 5-38. It
is preferred that the Her2 binding proteins of the invention
comprise amino acid sequences that exhibit at least 85% or at least
87% or at least 91% or at least 94% or at least 96% sequence
identity to one or more of the amino acid sequences of SEQ ID NO:
5-38. FIG. 1 shows examples for Her2 binding proteins. In further
embodiments, the Her2 binding protein based on SEQ ID NO: 1
comprises an insertion of amino acids within a natural loop region,
preferably within the first loop of the N-terminal part, in
addition to the substitutions in positions 62, 63, 64, 65, 66 of
SEQ ID NO: 1 and possibly further 1, 2, 3, 4, 5, or 6
modifications, for example in positions 2, 4, 6, or 8. A preferred
Her2 binding protein based on SEQ ID NO: 1 has substitutions in
amino acid region 62-66 of SEQ ID NO: 1 combined with an insertion
of 2-10 amino acids, preferably 4-9 amino acids, even more
preferred 6, 7, 8, or 9 amino acids, in a natural loop region of
said SEQ ID NO: 1, preferably in region 8-11, more preferably
between position 9 and 10 corresponding to SEQ ID NO: 1. For
example, Her2 binding Affilin-144567 (SEQ ID NO: 39) has an
insertion of 6 amino acids (PYETQV, SEQ ID NO: 42) at position 9 of
SEQ ID NO: 1 in addition to substitutions in positions 2, 4, 6, 62,
63, 64, 65, 66 SEQ ID NO: 1 (2R, 4G, 6G, 62R, 63F, 64W, 65K, 66K).
Her2 binding Affilin-143692 (SEQ ID NO: 40) has an insertion of 9
amino acids (AGNPSHMHH, SEQ ID NO: 43) at position 9 of SEQ ID NO:
1 in addition to substitutions in positions 2, 4, 6, 62, 63, 64,
65, 66 of SEQ ID NO: 1 (2D, 4D, 6M, 62H, 63W, 64I, 65L, 66N). Her2
binding proteins of the invention comprise amino acid sequences
selected from the group consisting of SEQ ID NO: 39 and SEQ ID NO:
40. It is preferred that the Her2 binding proteins comprise amino
acid sequences that exhibit at least 85% or at least 87% or at
least 91% or at least 94% or at least 96% sequence identity to one
or more of the amino acid sequences of SEQ ID NOs: 39-40.
[0068] The further characterization of Her2 binding proteins can be
performed in the form of soluble proteins. The appropriate methods
are known to those skilled in the art or described in the
literature. The methods for determining the binding affinities are
known per se and can be selected for instance from the following
methods known in the art: Surface Plasmon Resonance (SPR) based
technology, Bio-layer interferometry (BLI), enzyme-linked
immunosorbent assay (ELISA), flow cytometry, fluorescence
spectroscopy techniques, isothermal titration calorimetry (ITC),
analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and
enhanced chemiluminescence (ECL). Some of the methods are described
in the Examples below.
[0069] For stability analysis, for example spectroscopic or
fluorescence-based methods in connection with chemical or physical
unfolding are known to those skilled in the art. Exemplary methods
for characterization of Her2 binding proteins are outlined in the
Examples section of this invention.
[0070] For example, the biochemical target binding analysis is
summarized in FIG. 2 and further described in the Examples. All
binding proteins of the invention have an affinity of less than 700
nM for Her2, as determined by SPR based technology. In an
embodiment of the first aspect, the Her2-binding protein has a
dissociation constant K.sub.D to human Her2 in the range between
0.01 nM and 700 nM, more preferably between 0.05 nM and 500 nM,
more preferably between 0.1 nM and 100 nM, more preferably between
0.1 nM and 20 nM, more preferably between 0.1 nM and 10 nM. The
dissociation constant K.sub.D can be determined by ELISA or by
surface plasmon resonance assays. Typically, the dissociation
constant K.sub.D is determined at 20.degree. C., 25.degree. C., or
30.degree. C. If not specifically indicated otherwise, the K.sub.D
values recited herein are determined at 25.degree. C. by surface
plasmon resonance.
[0071] In addition, temperature stability was determined by
differential scanning fluorimetry (DSF), as described in further
detail in the Examples and as shown in FIG. 2. In addition to
results shown in FIG. 2, solubility of at least 80% was confirmed
for all Her2 binding molecules by size exclusion chromatography; no
Her2 binding molecule of the invention shows aggregation. FIG. 3
shows binding kinetics for two different Her2 binding proteins.
Competitive binding experiments comparing Affilin molecules show
that the epitope that is bound by different Her2 binding proteins,
for example Affilin-142628 and Affilin-143692, is not identical or
non-overlapping (see FIG. 12).
[0072] These Her2 binding proteins do not compete for Her2 binding.
Further, Her2 binding proteins bind to different Her2 epitopes than
the monoclonal antibody Trastuzumab. FIG. 13 shows that
Affilin-142628 and Affilin-143692 bind to different or
non-overlapping Her2 epitopes than Trastuzumab. In addition,
Affilin-141926 (SEQ ID NO: 28), Affilin-141884 (SEQ ID NO: 38),
Affilin-141890 (SEQ ID NO: 30), and Affilin-141975 (SEQ ID NO: 37)
bind to different or non-overlapping epitopes of Her2 than
Trastuzumab (Table 2). The first K.sub.D shown in Table 2 shows
binding to Her2, the second K.sub.D in the Table 2 shows binding to
Her2 in the presence of Trastuzumab. Since both values are almost
identical, it can be concluded that Affilin-proteins bind to
different or non-overlapping epitops than Trastuzumab. In contrast,
similar or overlapping epitopes with Trastuzumab show
Affilin-141931 (SEQ ID NO: 27), Affilin-141912 (SEQ ID NO: 31), and
Affilin-141935 (SEQ ID NO: 32).
TABLE-US-00002 TABLE 2 Competition of Affilin-proteins with
Trastuzumab Affilin- K.sub.D (nM) K.sub.D (nM) 141884 4.9 4.6
141890 24.3 25.5 141926 6.5 8.9 141975 41.2 39.4
[0073] Additional functional characterization was performed by
cellular Her2 binding analysis with Her2 overexpressing cells, for
example SkBr3 cells and genetically engineered CHO-K1 cells.
Different concentrations of the Affilin molecules were tested. Her2
cell target binding was confirmed, as shown in FIGS. 4-9.
[0074] Furthermore, Affilin binding proteins show binding to Her2
on tumor tissue from cells of human origin (see FIG. 10 and FIG.
11). In particular and surprisingly, Affilin molecules show strong
binding to Her2 expressed on SKOV-3 tumor tissue. No binding was
observed on tissue from lung, liver, heart muscle, and ovary.
[0075] One embodiment of the invention covers a Her2 binding
protein of the invention and further at least one additional
protein or molecule. The additional protein can be a second binding
protein with identical or different specificity for an antigen as
the first binding protein. One embodiment of the invention covers a
fusion protein or a conjugate comprising an Affilin-antibody fusion
protein or conjugate, optionally further fused with or conjugated
to a moiety preferably selected from at least one member of the
groups (i), (ii) and (iii) consisting of (i) a moiety modulating
pharmacokinetics selected from a polyethylene glycol (PEG), a human
serum albumin (HSA), a human serum albumin, an albumin-binding
peptide, or an immunoglobulin (Ig) or Ig fragments, a
polysaccharide, and, (ii) a therapeutically active component,
optionally selected from a monoclonal antibody or a fragment
thereof, a cytokine, a chemokine, a cytotoxic compound, an enzyme,
or derivatives thereof, or a radionuclide, and (iii) a diagnostic
component, optionally selected from a fluorescent compound, a
photosensitizer, a tag, an enzyme or a radionuclide. The conjugate
molecule can be attached e.g. at one or several sites through a
peptide linker sequence or a carrier molecule.
[0076] Further conjugation with proteinaceous or non-proteinaceous
moieties to generate protein conjugates according to the invention
can be performed applying chemical methods well-known in the art.
In particular, coupling chemistry specific for derivatization of
cysteine or lysine residues is applicable. In case of the
introduction of non-natural amino acids further routes of chemical
synthesis are possible, e.g. "click chemistry" or aldehyde specific
chemistry and others.
[0077] Conjugates thus obtained can be selected from one or more of
the following examples: (i) conjugation of the protein via lysine
residues; (ii) conjugation of the protein via cysteine residues via
maleimide chemistry; in particular, cysteine residues can be
specifically introduced and can be located at any position suitable
for conjugation of further moieties, (iii) peptidic or
proteinogenic conjugations. These and other methods for covalently
and non-covalently attaching a protein of interest to other
functional components are well known in the art, and are thus not
described in further detail here.
[0078] A further embodiment relates to binding proteins according
to the invention, further comprising a moiety modulating
pharmacokinetics or biodistribution, preferably selected from PEG,
HSA, or an Ig or Ig fragments, for example an Fc fragment. Several
techniques for producing proteins with extended half-life are known
in the art.
[0079] The binding protein of the invention may also comprise a
second binding protein which comprises or consists of a monoclonal
antibody or fragment thereof. In one embodiment, the second binding
protein is a monoclonal antibody with specificity for EGFR. It was
surprisingly found that a bispecific binding molecule consisting of
an EGFR monoclonal antibody and a Her2-specific Affilin is able to
bind specifically to both EGFR and Her2. The EGFR binding level of
the fusion protein is surprisingly higher than the EGFR-binding
level of Cetuximab.
[0080] In some embodiments of the invention, bispecific binding
molecules are provided comprising polypeptides specifically binding
to Her2 and to EGFR simultaneously. FIG. 14 shows the simultaneous
binding of bispecific Affilin-antibody binding proteins to both
target antigens (Her2 and EGFR). FIG. 15 shows the flow cytometric
binding analysis of Affilin-antibody binding proteins (e.g.
C-terminal fusion to light chain; CL-141926, SEQ ID NO: 44) on Her2
overexpressing cells (FIG. 15a) and on EGFR overexpressing cells
(FIG. 15b). The figure shows the mean fluorescence intensity,
representing the concentration dependent binding of the
Affilin-antibody fusion protein to Her2 and to EGFR overexpressing
cells.
[0081] In a further aspect of the invention, a Her2 binding protein
or fusion protein or conjugate is used in medicine, in particular
in a method of medical treatment or diagnosis, preferably in
cancer. The membrane protein Her2 is known to be upregulated in
tumor cells, resulting in uncontrolled growth of tumor cells and in
the formation of metastases. New therapies for cancer patients
include an inhibition of Her2 by targeted therapeutics such as for
example the monoclonal antibodies Trastuzumab (Herceptin.RTM.) or
Pertuzumab (Perjeta.RTM.). T-DM1, an antibody-drug conjugate, is
highly effective against breast, uterine, and ovarian
carcinosarcoma overexpressing Her2.
[0082] Overexpression of Her2 has been described in a wide variety
of cancers. For example, overexpression of Her2 occurs in
approximately 15% to 30% of breast cancers and 10% to 30% of
gastric/gastroesophageal cancers, and has also been observed in
other cancers like ovary, endometrium, bladder, lung colon, head
and neck. Thus, the pharmaceutical composition comprising the Her2
binding protein of the invention, can be used for treatment of
cancer in which Her2 is relevant for the development of the disease
including but not limited to particularly breast, ovarian, gastric,
but also in lung, head and neck, cervical, prostate, pancreas, and
others.
[0083] The compositions contain a therapeutically or diagnostically
effective dose of the Her2 binding protein of the invention. The
amount of protein to be administered depends on the organism to be
treated, the type of disease, the age and weight of the patient and
further factors known per se.
[0084] Some embodiments of the invention describe Her2 binding
proteins that bind with high affinity of at least 700 nM to the
extracellular domain of Her2 but have no or only weak cellular
binding. Such Her2 binding proteins are particularly useful for
certain medical applications requiring a differentiation of
Her2-binding proteins between soluble and cell-bound receptor.
Soluble Her2 is often found in the blood of cancer patients. The
Her2 binding proteins that bind to soluble Her2 (as for example in
Biacore assays) but not to cell-bound receptors can be used for
diagnostic applications where soluble Her2 is a predictive
biomarker for disease progression. Further, certain therapeutic
applications for Her2-binding Affilin proteins that only bind to
soluble Her2 can be useful, in particular in combination with a
therapeutic antibody that binds soluble and cell bound receptor
receptor (e.g. Trastuzumab). In this case, the Affilin would
preferably bind the soluble Her2 molecules, leaving more antibody
molecules available for the therapeutic intervention at the cell.
This opens the opportunity to lower the dose of the antibody known
for its cardiotoxic side effects. Examples of such Her2-binding
proteins are provided in this invention (e.g. Affilin-142465,
Affilin-142655, Affilin-142502, Affilin-141965, and
Affilin-144567).
[0085] The invention covers a pharmaceutical composition comprising
the Her2 binding protein, fusion protein or conjugate or the
nucleic acid molecule of the invention, the vector of the
invention, and/or the host cell or a virus and a pharmaceutically
acceptable carrier. The invention further covers a diagnostic agent
comprising the Her2 binding protein or conjugate or the nucleic
acid molecule of the invention, the vector of the invention, and/or
the host cell or non-human host with a diagnostically acceptable
carrier. The compositions contain a pharmaceutically or
diagnostically acceptable carrier and optionally can contain
further auxiliary agents and excipients known per se. These include
for example but are not limited to stabilizing agents,
surface-active agents, salts, buffers, coloring agents etc.
[0086] The pharmaceutical composition comprising the Her2 binding
protein can be in the form of a liquid preparation, a lyophilisate,
a cream, a lotion for topical application, an aerosol, in the form
of powders, granules, in the form of an emulsion or a liposomal
preparation. The compositions are preferably sterile, non-pyrogenic
and isotonic and contain the pharmaceutically conventional and
acceptable additives known per se. In addition, reference is made
to the regulations of the U.S. Pharmacopoeia or Remington's
Pharmaceutical Sciences, Mac Publishing Company (1990). In the
field of human and veterinary medical therapy and prophylaxis
pharmaceutically effective medicaments containing at least one Her2
binding protein in accordance with the invention can be prepared by
methods known per se. Depending on the galenic preparation these
compositions can be administered parentally by injection or
infusion, systemically, intraperitoneally, intramuscularly,
subcutaneously, transdermally or by other conventionally employed
methods of application. The type of pharmaceutical preparation
depends on the type of disease to be treated, the route of
administration, the severity of the disease, the patient to be
treated and other factors known to those skilled in the art of
medicine.
[0087] In a still further aspect the invention discloses diagnostic
compositions comprising Her2 binding protein according to the
invention specifically binding specific targets/antigens or its
isoforms together with diagnostically acceptable carriers. Since
enhanced Her2 expression is correlated with tumor malignancy, it is
desirable to develop diagnostics for non-invasive imaging in order
to gain information about Her2 expression status in patients.
Furthermore, Her2 imaging could be useful for the assessment of the
response of a patient to a therapeutic treatment. For example,
using a protein of the invention labelled with a suitable
radioisotope or fluorophore can be used for non-invasive imaging to
determine the location of tumors and metastasis (for review see for
example Milenic et al. 2008 Cancer Biotherapy &
Radiopharmaceuticals 23: 619-631; Hoeben et al. 2011, Int. Journal
Cancer 129: 870-878). Due to their pharmacokinetic characteristics,
intact antibodies are not suitable for routine imaging. Due to
their small size and high affinity, radiolabelled or fluorescently
labelled fusion proteins of the invention are expected to be much
better suited for use as diagnostics for imaging.
[0088] It is expected that a protein of the invention can be
advantageously applied in therapy. In particular, the molecules are
expected to show superior tumor targeting effect and desired
biodistribution and thus, reduced side effects. Pharmaceutical
compositions of the invention may be manufactured in any
conventional manner.
[0089] The derivatization of ubiquitin to generate a ubiquitin
mutein that specifically binds to a particular target antigen has
been described in the art. For example, a library can be created in
which the sequence as shown in SEQ ID NO: 4 has been altered.
Preferably, the alterations comprise at least 12 amino acids
selected from positions R42, I44, H68, V70, R72, L73, R74, K82,
L84, Q138, K139, E140, S141, and T142 of di-ubiquitin (SEQ ID NO:
4). In other embodiments, a library can be created in which the
sequence as shown in SEQ ID NO: 1 has been altered at least at
amino acids located in positions 62, 63, 64, 65, 66 of SEQ ID NO: 1
in combination with an extension of 4-10 amino acids in the
N-terminal loop. Additional 1, 2, 3, 4, 5, or 6 amino acids can be
substituted to generate a protein with a novel binding ability to
Her2.
[0090] The step of modification of the selected amino acids is
performed according to the invention preferably on the genetic
level by random mutagenesis of the selected amino acids.
Preferably, the modification of ubiquitin is carried out by means
of methods of genetic engineering for the alteration of a DNA
belonging to the respective protein.
[0091] Preferably, the alteration is a substitution, insertion or
deletion as described in the art. The substitution of amino acid
residues for the generation of the novel binding proteins derived
from ubiquitin can be performed with any desired amino acid. This
is described in detail in EP1626985B1, EP2379581B1, and EP2721152,
which are incorporated herein by reference.
[0092] The substitution of amino acids for the generation of the
novel binding proteins based on SEQ ID NO: 4 or SEQ ID
[0093] NO: 1 can be performed with any desired amino acid. This is
described in detail for example in EP1626985B1 and EP2379581B1,
which are incorporated herein by reference. Assuming a random
distribution of the 20 natural amino acids at e.g. 14 positions
generates a pool of 20 to the power of 14 (20.sup.14) theoretical
ubiquitin muteins, each with a different amino acid composition and
potentially different binding properties. This large pool of genes
constitutes a library of different Affilin binding proteins.
[0094] By way of example, starting point for the mutagenesis can be
for example the cDNA or genomic DNA coding for proteins of SEQ ID
NOs: 4 and 1. Furthermore, the gene coding for the protein as shown
in SEQ ID NOs: 4 and 1 can also be prepared synthetically. The DNA
can be prepared, altered, and amplified by methods known to those
skilled in the art. Different procedures known per se are available
for mutagenesis, such as methods for site-specific mutagenesis,
methods for random mutagenesis, mutagenesis using PCR or similar
methods. All methods are known to those skilled in the art.
[0095] In a preferred embodiment of the invention the amino acid
positions to be mutagenized are predetermined. In each case, a
library of different mutants is generally established using methods
known per se. Generally, a pre-selection of the amino acids to be
modified can be performed based on structural information available
for the ubiquitin protein to be modified. The selection of
different sets of amino acids to be randomized leads to different
libraries.
[0096] The gene pool libraries obtained as described above can be
combined with appropriate functional genetic elements which enable
expression of proteins for selection methods such as display
methods. The expressed proteins are contacted according to the
invention with a target molecule to enable binding of the partners
to each other if a binding affinity exists. This process enables
identification of those proteins which have a binding activity to
the target molecule. See, for example, EP2379581B1, which is
herewith incorporated by reference.
[0097] Contacting according to the invention is preferably
performed by means of a suitable presentation and selection method
such as the phage display, ribosomal display, mRNA display or cell
surface display, yeast surface display or bacterial surface display
methods, preferably by means of the phage display method. For
complete disclosure, reference is made also to the following
references: Hoess, Curr. Opin. Struct. Biol. 3 (1993), 572-579;
Wells and Lowmann, Curr. Opin. Struct. Biol. 2 (1992), 597-604; Kay
et al., Phage Display of Peptides and Proteins-A
[0098] Laboratory Manual (1996), Academic Press. The methods
mentioned above are known to those skilled in the art. The library
can be cloned into a phagemid vector (e.g. pCD87SA (Paschke, M. and
W. Hohne (2005). "Gene 350(1): 79-88)). The library may be
displayed on phage and subjected to repeated rounds of panning
against the respective target antigen. Ubiquitin muteins from
enriched phage pools are cloned into expression vectors for
individual protein expression. Preferably, expression of the
ubiquitin mutein enables screening for specific binding proteins by
established techniques, such as ELISA on automated high-throughput
screening platforms. Identified clones with desired binding
properties are then sequenced to reveal the amino acid sequences of
Affilin molecules. The identified binding protein may be subjected
to further maturation steps, e.g. by generating additional
libraries based on alterations of the identified sequences and
repeated phage display, ribosomal display, panning and screening
steps as described above.
[0099] Her2 binding molecules of the invention may be prepared by
any of the many conventional and well known techniques such as
plain organic synthetic strategies, solid phase-assisted synthesis
techniques, fragment ligation techniques or by commercially
available automated synthesizers. On the other hand, they may also
be prepared by conventional recombinant techniques alone or in
combination with conventional synthetic techniques. Furthermore,
they may also be prepared by cell-free in-vitro
transcription/translation. Conjugates according to the present
invention may be obtained by combining compounds by chemical
methods, e.g. lysine or cysteine-based chemistry, as described
herein above.
[0100] According to another aspect of the invention, an isolated
polynucleotide encoding a binding protein of the invention is
provided. The invention also encompasses polypeptides encoded by
the polynucleotides of the invention. The invention further
provides an expression vector comprising the isolated
polynucleotide of the invention, and a host cell comprising the
isolated polynucleotide or the expression vector of the
invention.
[0101] For example, one or more polynucleotides which encode for a
Her2 binding protein of the invention may be expressed in a
suitable host and the produced binding protein can be isolated.
Vectors comprising said polynucleotides are covered by the
invention. In a further embodiment the invention relates to a
vector comprising the nucleic acid molecule of the invention. A
vector means any molecule or entity (e.g., nucleic acid, plasmid,
bacteriophage or virus) that can be used to transfer protein coding
information into a host cell.
[0102] The present invention furthermore relates to an isolated
cell comprising the nucleic acid molecule of the invention or the
vector of the invention. Suitable host cells include prokaryotes or
eukaryotes. Various mammalian or insect cell culture systems can
also be employed to express recombinant proteins.
[0103] The invention also relates in an embodiment to a host cell
or a non-human host carrying the vector of the invention. A host
cell is a cell that has been transformed, or is capable of being
transformed, with a nucleic acid sequence and thereby expresses a
gene of interest. The term includes the progeny of the parent cell,
whether or not the progeny is identical in morphology or in genetic
make-up to the original parent cell, so long as the gene of
interest is present. In accordance with the present invention, the
host may be a transgenic non-human animal transfected with and/or
expressing the proteins of the present invention. In a preferred
embodiment, the transgenic animal is a non-human mammal.
[0104] In another aspect, there is provided a method of producing
the binding protein of the invention, comprising the steps of a)
culturing the host cell of the invention under conditions suitable
for the expression of the binding protein and b) isolating the
produced binding protein. The invention also encompasses a binding
protein produced by the method of the invention. Suitable
conditions for culturing a prokaryotic or eukaryotic host are well
known to the person skilled in the art.
[0105] One embodiment of the present invention is directed to a
method for the preparation of a binding protein according to the
invention as detailed above, said method comprising the following
steps: (a) preparing a nucleic acid encoding a Her2 binding protein
according to any aspect of the invention; (b) introducing said
nucleic acid into an expression vector; (c) introducing said
expression vector into a host cell; (d) cultivating the host cell;
(e) subjecting the host cell to culturing conditions under which a
Her2 binding protein is expressed, thereby producing a Her2 binding
protein as described above; (f)optionally isolating the Her2
binding protein produced in step (e); and (g) optionally
conjugating the Her2 binding protein with further functional
moieties as described above.
[0106] Cultivation of cells and protein expression for the purpose
of protein production can be performed at any scale, starting from
small volume shaker flasks to large fermenters, applying
technologies well-known to any skilled in the art.
[0107] Following the expression of the ubiquitin protein modified
according to the invention, it can be further purified and enriched
by methods known per se. The selected methods depend on several
factors known per se to those skilled in the art, for example the
expression vector used, the host organism, the intended field of
use, the size of the protein and other factors.
[0108] In general, isolation of purified protein from the
cultivation mixture can be performed applying conventional methods
and technologies well known in the art, such as centrifugation,
precipitation, flocculation, different embodiments of
chromatography, filtration, dialysis, concentration and
combinations thereof, and others. Chromatographic methods are
well-known in the art and comprise without limitation ion exchange
chromatography, gel filtration chromatography (size exclusion
chromatography), or affinity chromatography.
[0109] For simplified purification the protein modified according
to the invention can be fused to other peptide sequences having an
increased affinity to separation materials. Preferably, such
fusions are selected that do not have a detrimental effect on the
functionality of the ubiquitin mutein or can be separated after the
purification due to the introduction of specific protease cleavage
sites. Such methods are also known to those skilled in the art.
EXAMPLES
[0110] The following Examples are provided for further illustration
of the invention. The invention is particularly exemplified by
particular modifications of di-ubiquitin (SEQ ID NOs: 4 or 48) or
wild type ubiquitin (SEQ ID NOs: 1 or 2) resulting in binding to
Her2. The invention, however, is not limited thereto, and the
following Examples merely show the practicability of the invention
on the basis of the above description. For a complete disclosure of
the invention reference is made also to the literature cited in the
application which is incorporated completely into the application
by reference.
Example 1
Identification of Binding Proteins
[0111] Library Construction and Cloning Two ubiquitin moieties each
comprising seven randomized amino acid positions were synthesized
by triplet technology (MorphoSys Slonomics, Germany) to achieve a
well-balanced amino acid distribution. A mixture of 19-amino acid
coding premade double-stranded triplets excluding cysteine was used
for the synthesis. Both ubiquitin moieties were directly linked
(without linker between the two ubiquitin moieties) in head to tail
orientation to result in a protein of 152 amino acids with 14
randomized amino acid positions. The sequence of di-ubiquitin with
14 randomized positions is shown in SEQ ID NO: 3:
TABLE-US-00003 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQXLXFAGKQL
EDGRTLSDYNIQKESTLXLXLXXXAAMQIFVXTXTGKTITLEVEPSDTIE
NVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIXXXXXLHLVLRLR AA.
[0112] The 14 randomized amino acids correspond to positions 42,
44, 68, 70, 72, 73, 74, 82, 84, 138, 139, 140, 141, and 142 of
di-ubiquitin. The sequence of di-ubiquitin is shown in SEQ ID NO:
4:
TABLE-US-00004 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQL
EDGRTLSDYNIQKESTLHLVLRLRAAMQIFVKTLTGKTITLEVEPSDTIE
NVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIQKESTLHLVLRLR AA.
[0113] The construct was ligated with a modified pCD87SA phagemid
(herein referred to as pCD12) using standard methods known to a
skilled person. The pCD12 phagemid comprises a modified torA leader
sequence (deletion of amino acid sequence QPAMA) to achieve protein
processing without additional amino acids at the N terminus.
Aliquots of the ligation mixture were used for electroporation of
Escherichia coli ER2738 (Lucigen). The library is referred to as
SPIF. Unless otherwise indicated, established recombinant genetic
methods were used, for example as described in Sambrook et al.
[0114] Target: Recombinant human Her2 -Fc Chimera was purchased
from R&D Systems. A DNA sequence encoding the extracellular
domain of human Her2 (uniprot Accession Number p004626; residues
1-652) was genetically fused with the Fc region of human IgG1 at
the C-terminus.
[0115] TAT Phage Display Selection. The SPIF library was enriched
against the given protein target Her2 using TAT phage display as
selection system. After transformation of competent bacterial
ER2738 cells (Lucigene) with phagemid pCD12 carrying the SPIF
library, phage amplification and purification was carried out using
standard methods known to a skilled person. For selection the
target protein was provided as Fc-fusion protein (Her2-Fc, R&D
Systems) immobilized on Dynabeads.RTM. Protein A or G. The target
concentration during phage incubation varied from 200 nM (first
round) to 50 nM (third round). Target phage complexes were
magnetically separated from solution and washed several times.
Target bound phages were eluted by trypsin. To deplete the phage
library for Fc-binding variants a preselection of phages with
immobilized Fc-fragment of IgG1 (Athens Research & Technology)
was performed prior to round two and three.
[0116] To identify target specific phage pools, eluted and
reamplified phages of each selection round were analysed by phage
pool ELISA. Wells of a medium binding microtiter plate (Greiner
bio-one) were coated with Her2-Fc (2.5 .mu.g/ml) and Fc-fragment of
IgG1 (5 pg/ml), respectively. Bound phages were detected using
.alpha.-M13 HRP-conjugated antibody (GE Healthcare). Eluted and
reamplified phages of round three showed specific binding to the
target and were used subsequently for pool maturation by error
prone PCR. Isolated phagemid pools served as template for error
prone PCR (GeneMorph II Random Mutagenesis Kit, Agilent
Technologies). The amplified pool of SPIF variants carrying now
additional substitutions compared to the library positions was
recloned into phagemid pCD12 and transformed into ER2738 for phage
amplification and purification. The phages were again subjected to
two rounds of panning as described above. The target was employed
at a concentration of 5 nM and 1 nM in round one and two,
respectively. For both rounds a preselection with Fc-fragment of
IgG1 was performed. To analyze the matured and selected pools for
specific target binding a phage pool ELISA was performed as
described above.
[0117] Cloning of Target Binding Phage Pools into an Expression
Vector. Upon completion of the selection procedure the target
specific DNA pools of maturation selection round one and two were
amplified by PCR according to methods known in the art, cut with
appropriate restriction nucleases and ligated into a derivative of
the expression vector pET-28a (Merck, Germany) comprising a
Strep-Tag II (IBA GmbH).
[0118] Single Colony Hit Analysis. After transformation of BL21
(DE3) cells (Merck, Germany) kanamycin-resistant single colonies
were grown. Expression of the target-binding modified ubiquitin
variants was achieved by cultivation in 384 well plates (Greiner
BioOne) using auto induction medium (Studier, 2005). Cells were
harvested and subsequently lysed chemically or enzymatically by
BugBuster reagent (Novagen) or mechanically by freeze/thaw cycles,
respectively. After centrifugation the resulting supernatants were
screened by ELISA with immobilized target on highbind 384
micrrotiter plates (Greiner BioOne). Detection of bound protein was
achieved by Strep-Tactin.RTM. HRP Conjugate (IBA GmbH) in
combination with TMB-Plus substrate (Biotrend, Germany). The
reaction was stopped by addition of 0.2 M H.sub.2SO.sub.4 solution
and measured in a plate reader at 450 nm versus 620 nm.
Example 2
Expression and Purification of Her2-Binding Proteins
[0119] Affilin molecules were cloned to an expression vector using
standard methods known to a skilled person, purified and analyzed
as described below. All Affilin proteins were expressed and highly
purified by affinity chromatography and gel filtration. After
affinity chromatography purification a size exclusion
chromatography (SE HPLC or SEC) has been performed using an Akta
system and a Superdex.TM. 200 HiLoad 16/600 column (GE Healthcare).
The column has a volume of 120 ml and was equilibrated with 2 CV.
The samples were applied with a flow rate of 1 ml/min purification
buffer B. Fraction collection starts as the signal intensity
reaches 10 mAU. Following SDS-PAGE analysis positive fractions were
pooled and their protein concentrations were measured.
[0120] Further analysis included SDS-PAGE, SE-HPLC and RP-HPLC.
Protein concentrations were determined by absorbance measurement at
280 nm using the molar absorbent coefficient. RP chromatography (RP
HPLC) has been performed using a Dionex HPLC system and a Vydac
214M554 C4 (4.6.times.250 mm, 5 .mu.m, 300 .ANG.) column (GE
Healthcare).
Example 3
Solubility Analysis of Her2 Binding Proteins
[0121] Supernatants and resuspended pellets were analyzed by NuPage
Novex 4-12% Bis-Tris SDS gels and stained with Coomassie. Proteins
were recovered from the pellets by addition of 8 M urea. Her2
binding proteins displayed a high solubility of at least 80%
soluble (SEQ ID NO: 5, 27, 30, 37, 38), at least 90% soluble (SEQ
ID NOs: 6, 20, 23, 28, 34), at least 95% soluble expression (SEQ ID
NOs: 7, 9, 10, 11, 22, 29), 100% soluble (SEQ ID NOs: 8, 12, 13,
14, 15, 16, 17, 18, 19, 21, 25, 26, 33, 35, 36).
Example 4
Her2 Binding Proteins are Stable at High Temperatures
[0122] Thermal stability of the binding proteins of the invention
was determined by Differential Scanning Fluorimetry (DSF). Each
probe was transferred at concentrations of 0.1 .mu.g/.mu.L to a
MicroAmp Optical 384-well plate well plate, and SYPRO Orange dye
was added at suitable dilution. A temperature ramp from 25 to
95.degree. C. was programmed with a heating rate of 1.degree. C.
per minute (ViiA-7 Applied Biosystems). Fluorescence was constantly
measured at an excitation wavelength of 520 nm and the emission
wavelength at 623 nm (ViiA-7, Applied Biosystems). The midpoints of
transition for the thermal unfolding (Tm, melting points) are shown
for selected variants in FIG. 2. Her2 binding proteins of the
invention have similar melting temperatures. The stability of all
binding proteins is comparable to the stability of the control
proteins.
Example 5
Analysis of Her2 Binding Proteins (Surface Plasmon Resonance,
SPR)
[0123] A CM5 sensor chip (GE Healthcare) was equilibrated with SPR
running buffer. Surface-exposed carboxylic groups were activated by
passing a mixture of EDC and NHS to yield reactive ester groups.
700-1500 RU Her2-Fc(on-ligand) were immobilized on a flow cell,
IgG-Fc (off- ligand) was immobilized on another flow cell at a
ratio of 1:3 (hIgG-Fc:Target) to the target. Injection of
ethanolamine after ligand immobilization was used to block
unreacted NHS groups. Upon ligand binding, protein analyte was
accumulated on the surface increasing the refractive index. This
change in the refractive index was measured in real time and
plotted as response or resonance units (RU) versus time. The
analytes were applied to the chip in serial dilutions with a flow
rate of 30 .mu.l/min. The association was performed for 30 seconds
and the dissociation for 60 seconds. After each run, the chip
surface was regenerated with 30 .mu.l regeneration buffer and
equilibrated with running buffer. A dilution series of Trastuzumab
served as positive control, whereas a dilution series of unmodified
di-ubiquitin represents the negative control. The control samples
were applied to the matrix with a flow rate of 30 .mu.l/min, while
they associate for 60 seconds and dissociate for 120 seconds.
Regeneration and re-equilibration were performed as previously
mentioned. Binding studies were carried out by the use of the
Biacore 3000 (GE Healthcare); data evaluation was operated via the
BlAevaluation 3.0 software, provided by the manufacturer, by the
use of the Langmuir 1:1 model (RI=0). Results of binding to Her2
are shown in FIG. 2. Evaluated dissociation constants (K.sub.D)
were standardized against off-target and indicated.
Example 6
Functional Characterization: Binding to Cell Surface Expressed Her2
(Flow Cytometry)
[0124] Flow cytometry was used to analyze the interaction of Her2
binding proteins with surface-exposed Her2. Her2 overexpressing
human mammary gland adenocarcinoma-derived SkBr3 cells, Her2
overexpressing transfected CHO-K1 cells (chinese hamster ovary
cells), Her2 non-expressing human embryonic kidney cell line
HEK/293 and empty vector control CHO-K1 cells were used. Results
are summarized in FIGS. 4 to 8.
[0125] Cells were trypsinized and resupended in medium containing
FCS, washed and stained in pre-cooled FACS blocking buffer. A cell
concentration of 2.times.10.sup.6cells/ml was prepared for cell
staining and filled into a 96 well plate (Greiner) in triplicate
for each cell line.
[0126] Different concentrations of Affilin proteins were added to
Her2 overexpressing and control cells in several experiments. 50 nM
of each Affilin was tested on SkBr3 and Her2-negative HEK/293-cells
(FIGS. 4A and 4B). A dilution series from 333 nM to 5.6 nM (FIG. 5)
and a dilution series from 100 nM to 0.06 pM (FIG. 8) were added to
SkBr3-cells. On Her2-overexpressing CHO-K1 cells and the
Her2-negative CHO-K1-pEntry cell line Affilin concentrations of 500
nM to 0.5 nM (FIG. 6 and FIG. 7) were tested. After 45 min the
supernatants were removed and 100 .mu.l/well rabbit anti-Strep-Tag
antibody (obtained from GenScript; A00626), 1:300 diluted in FACS
blocking buffer were added. After removal of the primary antibody
goat anti-rabbit IgG Alexa Fluor 488 antibody (obtained from
Invitrogen; A11008) was applied in a 1:1000 dilution. Flow
cytometry measurement was conducted on the Guava easyCyte 5HT
device from Merck-Millipore at excitation wavelength 488 nm and
emission wavelength 525/30 nm. Results on SkBr3 for binding of
Affilin-141884, Affilin-141890, Affilin-141912, Affilin-141926,
Affilin-141931, Affilin-141935, Affilin-141965, Affilin-141975
(FIG. 4A), Affilin-142418, Affilin-142437, Affilin-142465,
Affilin-142502, Affilin-142609, Affilin-142618, Affilin-142620,
Affilin-142627, Affilin-142628, Affilin-142654, Affilin-142655,
Affilin-142672, Affilin-141884 (FIG. 4B) and Affilin-141926 and
Affilin-141890 (FIG. 5) are shown. Results on CHO-K1-Her2 cells for
binding of Affilin-142628, Affilin-142654, Affilin-141884,
Affilin-142627, Affilin-144631, Affilin-144632, Affilin-144633,
Affilin-144634, Affilin-144635, Affilin-144636, Affilin-144637,
Affilin-144567, and Affilin-142502 are depicted in FIG. 6a-c.
Concentration dependent binding of (50 nM to 0.5 nM) Affilin-142628
to CHO-K1-Her2 cells and CHO-K1-pEntry cells is shown in FIG. 7.
Comparable amounts of di-ubiquitin (139090) were used as negative
control in the experiments where applicable (e.g. in experiments as
shown in FIGS. 4A, 4B, 6A, 6B, and 7).
[0127] However, Affilin-142465 (SEQ ID NO: 33), Affilin-142655 (SEQ
ID NO: 34), Affilin-142502 (SEQ ID NO: 49), and Affilin-141965 (SEQ
ID NO: 50) showed only weak binding to Her2 overexpressing SkBr3
cells. SEQ ID NOs: 34, 35, 49 and 50 have at least the following
substitutions: 42S, 44V, 68Y, 70Y, 72F, 73S, 82L, 84D, 138R, 139G,
140W, 142L (of di-ubiquitin). Affilin-142418 and Affilin-142655
have further two substitutions (K631, Q78R) or further four
substitutions (Q31L, D58V, Q78R, P95S), respectively. Her2 binding
proteins that bind with high affinity of at least 700 nM to the
extracellular domain of Her2 but without or with low cellular
binding are particularly useful for certain applications, e.g. for
certain diagnostic or therapeutic applications that require
Her-binding proteins with high affinity only for soluble Her2, but
not for cellular Her2. Affilin-142418 shows binding to Her2
overexpressing SkBr3 cells (FIG. 4b, FIG. 9).
Example 7
Binding to Cell Surface Expressed Her2 (Immunocytochemistry and
Fluorescence Microscopy)
[0128] Binding of proteins of the invention on cells exogenously
expressing human Her2 was confirmed. 50 nM of Affilin-141884,
Affilin-142628, Affilin-141926, Affilin-144637, Affilin-142418 and
Affilin-144567 were tested on Her2-expressing SkBr3 cells and the
negative control cell line HEK/293. Di-ubiquitin (139090) was used
as negative control and 10nM Trastuzumab served as positive
control. Cells were seeded with a concentration of
1.times.10.sup.5cells/ml in Lab-Tek.RTM. Chamber-Slides
(Sigma-Aldrich). After cultivation over 72 h the cells were fixed
with methanol (5 min., 20.degree. C.), followed by blocking (5%
Fetal Horse Serum in PBS, 1 h) and incubation with 50 nM Affilin
for 45 min at rt. Affilin binding was detected by an incubation of
rabbit-anti-Strep-Tag-antibody (1:500) for 1h and subsequent
incubation with anti-rabbit-IgG-Alexa488-antibody (1:1000) for 1 h.
Trastuzumab binding were proved with
anti-human-IgG-Alexa488-antibody (1:1000). The nuclei were stained
with 4 .mu.g/mI DAPI. All incubation steps were done at room
temperature. FIG. 9A shows strong binding of Affilin-141884,
Affilin-142628, Affilin-141926, Afflin-144637 and Affilin-142418.
Weak or negative binding of Affilin-144567 and di-ubiquitin is
shown in FIG. 9B. Comparable binding was detected with Trastuzumab.
No non-specific binding was observed on Her2-negative cell line
HEK/293.
Example 8
Functional Characterization: Her2 Binding Proteins Bind to
Extracellular Her2 Expressed on Tumor Cells (Immunohistochemistry
and Fluorescence Microscopy)
[0129] Cryo-tissue sections of SKOV-3-tumor, lung, liver, heart
muscle and ovary were used to analyze the binding proteins of the
invention. Tissue slices were fixed with ice-cold Acetone for 10
min, followed by blocking and incubation with different
concentrations (20 nM and 50 nM) of Affilin-141884 and
Affilin-142628 and an equal amount of di-ubiquitin clone 139090 as
negative or 10 nM Trastuzumab as positive control for 1 h. After
washing with PBS the tissue was incubated with rabbit
anti-Strep-Tag antibody (1:500) for 1 h at room temperature,
followed by an incubation with goat anti-rabbit Alexa488 (1:1000)
or goat anti-human IgG Alexa594 (1:1000) as secondary antibody for
Trastuzumab. Nuclei of cells were visualized with DAPI. Chamber
slides were dissembled and the glass slides were covered with
Mowiol and a cover glass. Slices were imaged at a Zeiss Axio
Scope.A1 microscope and images were processed using standard
software packages. FIGS. 10 and 11 show the specific binding of
Affilin-141884 and Affilin-142628 on SKOV-3-tumor-slices compared
to the non-binding protein clone 139090. No binding on lung tissue
was obtained (FIG. 11). Slices of further tissues (liver, heart
muscle and ovarian tissue) were tested; no specific staining was
observed.
Example 9
Competition Analysis that Her2 Binding Affilin Molecules Bind to
Other Epitope Than anti-Her2 Monoclonal Antibody Trastuzumab
[0130] Affilin proteins that bind to different Her2 epitopes of
particular Trastuzumab can be useful in certain medical
embodiments. To investigate whether the isolated Her2 binding
proteins of the invention can compete with the anti-Her2 monoclonal
antibody Trastuzumab, the following assay was performed: Her2 (from
Acrobiosystems) was immobilized on a CM5 Biacore chip using NHS/EDC
chemistry resulting in 1000 response units (RU). In a first
experiment, all variants were injected at one defined concentration
(2.5 pM) at a flow of 30 .mu.l/min PBST 0.005% Tween 20 (FIG. 12).
In the second experiment, the same flow channel was first
pre-loaded with Trastuzumab (200 nM) until the chip surface was
saturated (FIG. 13). After loading Trastuzumab, the variants were
identically applied as in experiment 1 (2.5 pM). For better
clarification both sensogram traces were aligned at the last
injected Her2 binding protein.
[0131] It was demonstrated that the binding of a Her2 binding
protein was not influenced by the presence of Trastuzumab. Thus, no
competition was observed, meaning that Trastuzumab and Her2 binding
proteins of the invention bind to different or non-overlapping
Her2-epitopes, i.e. to different surface exposed amino acids, than
Trastuzumab (see FIG. 13). This was observed for Affilin-142628
(SEQ ID NO: 19), Affilin-143692 (SEQ ID NO: 39), Affilin-141926
(SEQ ID NO: 28), Affilin-141884 (SEQ ID NO: 38), Affilin-141890
(SEQ ID NO: 30), and Affilin-141975 (SEQ ID NO: 37). These binding
proteins might be particularly useful in cancer treatments with
reported primary and acquired resistance to Trastuzumab.
Affilin-141931 (SEQ ID NO: 27), Affilin-141912 (SEQ ID NO: 31), and
Affilin-141935 (SEQ ID NO: 32) bind to identical or overlapping
Her2-epitopes as Trastuzumab.
Example 10
Binding Analysis of Bispecific Fusion Proteins of Her2 Binding
Protein and EGFR-antibody Cetuximab
[0132] A Her2 binding protein was linked to the C- or N-terminus of
the light chain or heavy chain of the anti-EGFR monoclonal antibody
Cetuximab. Fusion proteins were generated by fusing a Her2 binding
protein (for example, Affilin-141926) to the N-terminus of the
heavy chain of the anti EGFR antibody Cetuximab (referred to as
NH-141926; SEQ ID NO: 47), to the C- terminus of the heavy chain of
the anti EGFR antibody Cetuximab (referred to as CH-141926; SEQ ID
NO: 45), to the N-terminus of the light chain of the anti EGFR
antibody Cetuximab (referred to as NL-141926; SEQ ID NO: 46), and
to the C- terminus of the light chain of the anti-EGFR antibody
Cetuximab (Erbitux.RTM.; referred to as CL-141926; SEQ ID NO: 44)
respectively. The first up to 20 amino acids of SEQ ID NOs: 44-47
are signal sequences. The cDNA encoding the fusion proteins were
transiently transfected into FreeStyle.TM. 293-F cells and
expressed in serum-free/animal component-free media. Expression was
confirmed by Western Blot analysis. Fusion proteins were purified
from the supernatants by Protein A affinity chromatography
(GE-Healthcare cat no 17-0402-01) with an AKTAxpress.RTM. (GE
Healthcare). Further purification of the fusion proteins was
achieved by gel filtration. Further analysis included SDS-PAGE,
SE-HPLC and RP-HPLC. Thermal stability of the binding proteins of
the invention was determined by Differential Scanning Fluorimetry
as described above. The midpoint of transition for the thermal
unfolding (Tm, melting points) was determined for fusion proteins;
all fusions proteins have thermal stabilities between
T.sub.m=63.9.degree. C. and 67.9.degree. C. (see Tab. 3). The
stability of all binding proteins is comparable to the stability of
the control proteins.
TABLE-US-00005 TABLE 3 Midpoint of transition for the thermal
unfolding of binding proteins of the invention and of control
proteins Fusion protein or control Tm [.degree. C.] Cetuximab 69.0
CH-141926 67.4 CL-141926 63.9 NH-141926 67.5 NL-141926 67.9
[0133] Binding studies were carried out by the use of the
Biacore.RTM. 3000 (GE Healthcare) as described above and as shown
in FIG. 14. Further, FACS analysis of binding of the fusion
proteins to the target receptors expressed individually in CHO-K1
cells confirmed cell binding (FIG. 15). Results are summarized in
Tab. 4 and Tab. 5.
TABLE-US-00006 TABLE 4 Affinity data for
Her2-ubiquitin-mutein-Cetuximab binding proteins of the invention
for EGFR (Biacore) Fusion protein k.sub.on [M.sup.-1 .times.
s.sup.-1] k.sub.off [s.sup.-1] K.sub.D [M] Cetuximab 6.21 .times.
10.sup.5 6.29 .times. 10.sup.-4 1.01 .times. 10.sup.-9 CH-ubiquitin
7.28 .times. 10.sup.5 6.72 .times. 10.sup.-4 .sup. 9.23 .times.
10.sup.-10 CH-141926 4.66 .times. 10.sup.5 1.65 .times. 10.sup.-4
.sup. 3.53 .times. 10.sup.-10 CL-ubiquitin 7.96 .times. 10.sup.5
7.12 .times. 10.sup.-4 .sup. 8.95 .times. 10.sup.-10 CL-141926 5.84
.times. 10.sup.5 5.91 .times. 10.sup.-4 1.01 .times. 10.sup.-9
NH-ubiquitin 3.02 .times. 10.sup.5 6.5 .times. 10.sup.-4 2.15
.times. 10.sup.-9 NH-141926 3.79 .times. 10.sup.5 4.12 .times.
10.sup.-4 1.09 .times. 10.sup.-9 NL-ubiquitin 2.79 .times. 10.sup.5
1.54 .times. 10.sup.-5 .sup. 5.52 .times. 10.sup.-11 NL-141926 1.08
.times. 10.sup.5 1.72 .times. 10.sup.-4 1.59 .times. 10.sup.-9
TABLE-US-00007 TABLE 5 Affinity data for
Her2-ubiquitin-mutein-Cetuximab binding proteins of the invention
for Her2 (Biacore) Fusion protein k.sub.on [M.sup.-1 .times.
s.sup.-1] k.sub.off [s.sup.-1] K.sub.D [M] CH-141926 7.53 .times.
10.sup.4 4.96 .times. 10.sup.-4 6.58 .times. 10.sup.-9 CL-141926
3.11 .times. 10.sup.5 4.46 .times. 10.sup.-4 1.43 .times. 10.sup.-9
NH-141926 9.03 .times. 10.sup.4 3.05 .times. 10.sup.-4 3.37 .times.
10.sup.-9 NL-141926 8.23 .times. 10.sup.4 2.36 .times. 10.sup.-4
2.87 .times. 10.sup.-9
[0134] It is known that a co-expression of the receptor proteins
EGFR and Her2 on various forms of cancer (e.g. breast, colorectal
and prostate cancer) is associated with poor prognosis for the
patients. In FIG. 14, a Biacore chip with immobilized extracellular
domain Her2-Fc was used. The bispecific fusion proteins were
injected, after 350 sec, extracellular domain EGFR-Fc was injected
at concentrations between 100 nM and 25 nM in 1:2 dilution series.
FIG. 14 shows the simultaneous binding of bispecific
Affilin-antibody fusion proteins to both targets. This effect might
increase the selectivity and efficiency of a product comprising a
bispecific EGFR-Her2-fusion protein of the invention in therapeutic
applications in molecular imaging. In FIG. 15 the binding of the
two interaction moieties to their respective targets is shown on
Her2 (FIG. 15a) and EGFR (FIG. 15b) overexpressing CHO cells by
flow cytometry measurements for variant CL 141926.
Sequence CWU 1
1
50176PRTArtificial sequenceArtificially synthesized wildtype
ubiquitin 1Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr
Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg
Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu
Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val
Leu Arg Leu Arg Gly Gly 65 70 75 276PRTArtificial
sequenceArtificially synthesized ubiquitin reference 2Met Gln Ile
Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25
30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys
35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln
Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Ala Ala 65
70 75 3152PRTArtificial sequenceArtificially synthesized ubiquitin
referencemisc_feature(42)..(42)Xaa can be any naturally occurring
amino acidmisc_feature(44)..(44)Xaa can be any naturally occurring
amino acidmisc_feature(68)..(68)Xaa can be any naturally occurring
amino acidmisc_feature(70)..(70)Xaa can be any naturally occurring
amino acidmisc_feature(72)..(74)Xaa can be any naturally occurring
amino acidmisc_feature(82)..(82)Xaa can be any naturally occurring
amino acidmisc_feature(84)..(84)Xaa can be any naturally occurring
amino acidmisc_feature(138)..(142)Xaa can be any naturally
occurring amino acid 3Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys
Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp
Gln Gln Xaa Leu Xaa Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu
Xaa Leu Xaa Leu Xaa Xaa Xaa Ala Ala Met Gln Ile Phe 65 70 75 80 Val
Xaa Thr Xaa Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90
95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile
100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu
Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Xaa Xaa Xaa
Xaa Xaa Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
4152PRTArtificial sequenceArtificially synthesized di-ubiquitin
clone 139090 4Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys
Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr
Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu
Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105
110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp
115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr
Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
5152PRTArtificial sequenceArtificially synthesized Affilin-142437
5Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asn Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Thr Pro Pro Asp Gln Gln Thr Leu Ala Phe
Ala Gly Lys 35 40 45 His Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp
Asn Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys
Asn Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 6152PRTArtificial
sequenceArtificially synthesized Affilin-142672 6Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Thr Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Leu Thr Asp Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Arg Gly Trp Ser Leu Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 7152PRTArtificial sequenceArtificially
synthesized Affilin-144637 7Met Gln Ile Phe Val Lys Thr Leu Thr Gly
Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Thr Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro
Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu Tyr
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr
Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70 75 80
Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Asp Val Glu Pro Ser 85
90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly
Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln
Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu
Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145
150 8152PRTArtificial sequenceArtificially synthesized
Affilin-144636 8Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Thr Glu Asn Val Lys
Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln
Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu Tyr Gly Arg Thr
Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu
Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr Thr
His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105
110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp
115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile
Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
9152PRTArtificial sequenceArtificially synthesized Affilin-144635
9Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe
Val Gly Met 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp
Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys
Thr Ile Thr Leu Asp Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Met Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 10152PRTArtificial
sequenceArtificially synthesized Affilin-144634 10Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu
Asp Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Met Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 11152PRTArtificial sequenceArtificially
synthesized Affilin-144633 11Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu
Tyr Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Asp Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 12152PRTArtificial sequenceArtificially synthesized
Affilin-144632 12Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Thr Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp
Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr
Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100
105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala
Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
13152PRTArtificial sequenceArtificially synthesized Affilin-144631
13Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe
Val Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp
Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys
Thr Ile Thr Leu Asp Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 14152PRTArtificial
sequenceArtificially synthesized Affilin-142679 14Met Gln Ile Phe
Val Arg Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 15152PRTArtificial sequenceArtificially
synthesized Affilin-142658 15Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Thr Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro
Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu Gln 130
135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 16152PRTArtificial
sequenceArtificially synthesized Affilin-142654 16Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 17152PRTArtificial sequenceArtificially
synthesized Affilin-142653 17Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Ser Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 18152PRTArtificial sequenceArtificially synthesized
Affilin-142645 18Met Gln Ile Phe Val Lys Thr Leu Thr Asp Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Thr Leu Ala Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp
Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr
Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100
105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp 115 120 125 Gly Arg Thr Leu Thr Asp Tyr Asn Ile Ser Glu Trp Ala
Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
19152PRTArtificial sequenceArtificially synthesized Affilin-142628
19Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Thr Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe
Val Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp
Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys
Thr Ile Thr Leu Asp Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 20152PRTArtificial
sequenceArtificially synthesized Affilin-142620 20Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Val Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 21152PRTArtificial sequenceArtificially
synthesized Affilin-142618 21Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Lys Gln Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Gln Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 22152PRTArtificial sequenceArtificially synthesized
Affilin-142617 22Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Thr Leu Ala Phe Val Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp
Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr
Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100
105 110 Pro Ser Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp 115 120 125 Gly Arg Ala Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala
Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
23152PRTArtificial sequenceArtificially synthesized Affilin-142609
23Met Gln Ile Phe Val Lys Thr Leu Thr Gly Asn Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe
Val Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp
Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys
Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Pro Arg Leu Arg Ala Ala 145 150 24152PRTArtificial
sequenceArtificially synthesized Affilin-142451 24Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Val Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Thr 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Asn Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 25152PRTArtificial sequenceArtificially
synthesized Affilin-142441 25Met Gln Ile Phe Val Lys Thr Leu Thr
Asn Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Ala Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 26152PRTArtificial sequenceArtificially synthesized
Affilin-142439 26Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Thr Leu Ala Phe Ala Gly Lys 35 40 45 Gln Leu Glu Tyr Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp
Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70 75 80 Val Thr
Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100
105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala
Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
27152PRTArtificial sequenceArtificially synthesized Affilin-141931
27Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe
Val Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp
Tyr Ala Ala Met Gln Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys
Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 28152PRTArtificial
sequenceArtificially synthesized Affilin-141926 28Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe Ala Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met
Arg Ile Phe 65 70 75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 29152PRTArtificial sequenceArtificially
synthesized Affilin-141869 29Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Ala Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Gln Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu
Arg Ala Ala 145 150 30152PRTArtificial sequenceArtificially
synthesized Affilin-141890 30Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Ala Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Gln Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 31152PRTArtificial sequenceArtificially synthesized
Affilin-141912 31Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Val Pro Pro Asp Gln
Gln Thr Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Phe
Leu Tyr Leu Tyr Val Ser Ala Ala Met Gln Ile Phe 65 70 75 80 Val Leu
Thr Asp Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100
105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Glu Leu Trp Arg
Asn Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
32152PRTArtificial sequenceArtificially synthesized Affilin-141935
32Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Asp Gly Ile Pro Pro Asp Gln Gln Thr Leu Thr Phe
Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Tyr Leu Tyr Leu Gly Ile
Ser Ala Ala Met Gln Ile Phe 65 70 75 80 Val Ile Thr Ser Thr Gly Lys
Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Glu Leu Trp Arg Asn Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 33152PRTArtificial
sequenceArtificially synthesized Affilin-142465 33Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Leu Leu Ser Phe Ala Gly Lys 35
40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu His Leu Trp Leu Gly Val Arg Ala Ala Met
Gln Ile Phe 65 70 75 80 Val Asn Thr Glu Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 34152PRTArtificial sequenceArtificially
synthesized Affilin-142655 34Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Leu Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Ser Leu Val Phe Ala Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Val Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Tyr Leu Tyr Leu Phe Ser Arg Ala Ala Met Arg Ile Phe 65 70
75 80 Val Leu Thr Asp Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Arg Gly Trp Ser Leu Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 35152PRTArtificial sequenceArtificially synthesized
Affilin-142418 35Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Ser Leu Val Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Ile Glu 50 55 60 Ser Thr Leu Tyr
Leu Tyr Leu Phe Ser Arg Ala Ala Met Arg Ile Phe 65 70 75 80 Val Leu
Thr Asp Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100
105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Gly Trp Ser
Leu Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150
36152PRTArtificial sequenceArtificially synthesized Affilin-142627
36Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Val Glu 1
5 10 15 Val Glu Pro Cys Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Ser Leu Val Phe
Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu Tyr Leu Tyr Leu Phe Ser
Arg Ala Ala Met Arg Ile Phe 65 70 75 80 Val Leu Thr Asp Thr Gly Lys
Thr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135
140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 37152PRTArtificial
sequenceArtificially synthesized Affilin-141975 37Met Gln Ile Phe
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Ser Leu Val Phe Ala Gly Lys 35
40 45 Gln Leu Glu Tyr Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60 Ser Thr Leu Tyr Leu Tyr Leu Phe Ser Arg Val Ala Met
Arg Ile Phe 65 70 75 80 Val Leu Thr Asp Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Pro Asn 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Ser Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Ala Leu Ser
Asp Tyr Asn Ile Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu
Arg Leu Arg Ala Ala 145 150 38152PRTArtificial sequenceArtificially
synthesized Affilin-141884 38Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Arg Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro
Pro Asp Gln Gln Thr Leu Ala Phe Ala Gly Lys 35 40 45 Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser
Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met Arg Ile Phe 65 70
75 80 Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Ser Glu Trp Ala Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala
Ala 145 150 3982PRTArtificial sequenceArtificially synthesized LOOP
BINDER 1>144567 (6er LOOP) 39Met Gln Ile Phe Val Lys Thr Leu Thr
Pro Tyr Glu Thr Gln Val Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val
Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln
Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg 35 40 45 Leu Ile Trp
Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr
Asn Ile Arg Gly Gly Arg Phe Leu Trp Leu Lys Leu Lys Leu Arg 65 70
75 80 Ala Ala 4085PRTArtificial sequenceArtificially synthesized
LOOP BINDER 2>143692 (9er LOOP) 40Met Gln Ile Phe Val Lys Thr
Leu Thr Ala Gly Asn Pro Ser His Met 1 5 10 15 His His Gly Lys Thr
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile 20 25 30 Glu Asn Val
Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp 35 40 45 Gln
Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 50 55
60 Leu Ser Asp Tyr Asn Ile Asp Asp Met His Trp Leu Ile Leu Leu Leu
65 70 75 80 Asn Leu Arg Ala Ala 85 414PRTArtificial
sequenceArtificially synthesized general linker sequence 41Ser Gly
Gly Gly 1 426PRTArtificial sequenceArtifical insertion of 6 amino
acid in Affilin-144567 42Pro Tyr Glu Thr Gln Val 1 5
439PRTArtificial sequenceArtifical insertion of 9 amino acids in
Affilin-143692 43Ala Gly Asn Pro Ser His Met His His 1 5
44401PRTArtificial sequenceArtificially synthesized fusion protein
of Affilin SEQ ID NO 28 to c-terminus of light chain of Cetuximab
(refered to as CL-141926) 44Met Val Ser Thr Pro Gln Phe Leu Val Phe
Leu Leu Phe Trp Ile Pro 1 5 10 15 Ala Ser Arg Ser Asp Ile Leu Leu
Thr Gln Ser Pro Val Ile Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg
Val Ser Phe Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Gly Thr Asn
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro 50 55 60 Arg Leu
Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser 65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85
90 95 Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn
Asn 100 105 110 Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210
215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gln Ile
Phe Val Lys Thr 245 250 255 Leu Thr Gly Lys Thr Ile Thr Leu Glu Val
Glu Pro Ser Asp Thr Ile 260 265 270 Glu Asn Val Lys Ala Lys Ile Gln
Asp Lys Glu Gly Ile Pro Pro Asp 275 280 285 Gln Gln Thr Leu Ala Phe
Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr 290 295 300 Leu Ser Asp Tyr
Asn Ile Gln Lys Glu Ser Thr Leu Trp Leu Tyr Leu 305 310 315 320 Thr
Trp Tyr Ala Ala Met Arg Ile Phe Val Thr Thr His Thr Gly Lys 325 330
335 Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys
340 345 350 Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu 355 360 365 Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr
Leu Ser Asp Tyr 370 375 380 Asn Ile Ser Glu Trp Ala Ile Leu His Leu
Val Leu Arg Leu Arg Ala 385 390 395 400 Ala 45635PRTArtificial
sequenceArtificially synthesized fusion protein of Affilin SEQ ID
NO 28 to c-terminus of heavy chain of Cetuximab (refered to as
CH-141926) 45Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe
Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Gln Leu Lys Gln Ser Gly
Pro Gly Leu Val Gln 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Asn Tyr Gly Val His Trp
Val Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val
Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr 65 70 75 80 Pro Phe Thr
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val
Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr 100 105
110 Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp
115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr
Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225
230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345
350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 465 470
475 480 Gly Gly Ser Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile 485 490 495 Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys 500 505 510 Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln
Gln Thr Leu Ala Phe 515 520 525 Ala Gly Lys Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile 530 535 540 Gln Lys Glu Ser Thr Leu Trp
Leu Tyr Leu Thr Trp Tyr Ala Ala Met 545 550 555 560 Arg Ile Phe Val
Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu Val 565 570 575 Glu Pro
Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 580 585 590
Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln 595
600 605 Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp
Ala 610 615 620 Ile Leu His Leu Val Leu Arg Leu Arg Ala Ala 625 630
635 46401PRTArtificial sequenceArtificially synthesized fusion
protein of Affilin SEQ ID NO 28 to n-terminus of light chain of
Cetuximab (refered to as NL-141926) 46Met Val Ser Thr Pro Gln Phe
Leu Val Phe Leu Leu Phe Trp Ile Pro 1 5 10 15 Ala Ser Arg Ser Met
Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr 20 25 30 Ile Thr Leu
Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala 35 40 45 Lys
Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala 50 55
60 Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
65 70 75 80 Ile Gln Lys Glu Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr
Ala Ala 85 90 95 Met Arg Ile Phe Val Thr Thr His Thr Gly Lys Thr
Ile Thr Leu Glu 100 105 110 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 115 120 125 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Arg Leu Ile Trp Ala Gly Lys 130 135 140 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Ser Glu Trp 145 150 155 160 Ala Ile Leu
His Leu Val Leu Arg Leu Arg Ala Ala Gly Gly Gly Gly 165 170 175 Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Leu Leu Thr 180 185
190 Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe
195 200 205 Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp
Tyr Gln 210 215 220 Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys
Tyr Ala Ser Glu 225 230 235 240 Ser Ile Ser Gly Ile Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr 245 250 255 Asp Phe Thr Leu Ser Ile Asn
Ser Val Glu Ser Glu Asp Ile Ala Asp 260 265 270 Tyr Tyr Cys Gln Gln
Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala Gly 275 280 285 Thr Lys Leu
Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 290 295 300 Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 305 310
315 320 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys 325 330 335 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu 340 345 350 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu 355 360 365 Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr 370 375 380 His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu 385 390 395 400 Cys
47643PRTArtificial sequenceArtificially synthesized fusion protein
of Affilin SEQ ID NO 28 to n-terminus of heavy chain of Cetuximab
(refered to as NH-141926) 47Met Ala Val Leu Gly Leu Leu Phe Cys Leu
Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Met Gln Ile Phe Val
Lys Thr Leu Thr Gly Lys Thr Ile 20 25 30 Thr Leu Glu Val Glu Pro
Ser Asp Thr Ile Glu Asn Val Lys Ala Lys 35 40 45 Ile Gln Asp Lys
Glu Gly Ile Pro Pro Asp Gln Gln Thr Leu Ala Phe 50 55 60 Ala Gly
Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile 65 70 75 80
Gln Lys Glu Ser Thr Leu Trp Leu Tyr Leu Thr Trp Tyr Ala Ala Met 85
90 95 Arg Ile Phe Val Thr Thr His Thr Gly Lys Thr Ile Thr Leu Glu
Val 100 105 110 Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile
Gln Asp Lys 115 120 125 Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
Trp Ala Gly Lys Gln 130 135 140 Leu Glu Asp Gly Arg Thr Leu Ser Asp
Tyr Asn Ile Ser Glu Trp Ala 145 150 155 160 Ile Leu His Leu Val Leu
Arg Leu Arg Ala Ala Gly Gly Gly Gly Ser 165 170 175 Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln 180 185 190 Ser Gly
Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys 195 200 205
Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg 210
215 220 Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser
Gly 225 230 235 240 Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg
Leu Ser Ile Asn 245 250 255 Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
Lys Met Asn Ser Leu Gln 260 265 270 Ser Asn Asp Thr Ala Ile Tyr Tyr
Cys Ala Arg Ala Leu Thr Tyr Tyr 275 280 285 Asp Tyr Glu Phe Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser 290 295 300 Ala Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 305 310 315 320 Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 325 330
335 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
340 345 350 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr 355 360 365 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln 370 375 380 Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp 385 390 395 400 Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro 405 410 415 Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 420 425 430 Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 435 440 445 Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 450 455
460 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
465 470 475 480 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val 485 490 495 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 500 505 510 Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys 515 520 525 Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu 530 535 540 Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 545 550 555 560 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 565 570 575
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 580
585 590 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 595 600 605 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 610 615 620 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Trp Ser His Pro Gln 625 630 635 640 Phe Glu Lys 48152PRTArtificial
sequenceArtificially synthesized di-ubiquitin reference 48Met Gln
Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20
25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly
Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly
Gly Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys
Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln
Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr
Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His 130 135 140 Leu
Val Leu Arg Leu Arg Gly Gly 145 150 49152PRTArtificial
sequenceArtificially synthesized clone 142502 49Met Gln Ile Phe Val
Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro
Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys
Glu Gly Ile Pro Pro Asp Gln Gln Ser Leu Val Phe Ala Gly Lys 35 40
45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
50 55 60 Ser Thr Leu Tyr Leu Tyr Leu Phe Ser Arg Ala Ala Met Gln
Ile Phe 65 70 75 80 Val Leu Thr Asp Thr Gly Lys Thr Ile Thr Leu Glu
Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile
Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile
Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp
Tyr Asn Ile Arg Gly Trp Ser Leu Leu His 130 135 140 Leu Val Leu Arg
Leu Arg Ala Ala 145 150 50152PRTArtificial sequenceArtificially
synthesized clone 141965 50Met Gln Ile Phe Val Lys Thr Leu Thr Gly
Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro
Asp Gln Gln Ser Leu Val Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr
Leu Tyr Leu Tyr Leu Phe Ser Arg Ala Ala Met Gln Ile Phe 65 70 75 80
Val Leu Thr Asp Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 85
90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly
Ile 100 105 110 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Met Gln
Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Gly
Trp Ser Leu Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145
150
* * * * *
References